Transgenic mouse model for degeneration of type II collagen in joints

ABSTRACT

The present invention provides animal model systems for cartilage-degenerative disease, which comprise transgenic animals which can express recombinant matrix-degrading enzymes (MDEs), particularly matrix metalloproteinases (MMPs), in a temporally and spatially regulated manner. The invention also provides methods for producing phenotypic indicators of cartilage-degenerative disease in a mammal and methods for determining the potential of a composition to counteract cartilage-degenerative disease. The invention also provides isolated nucleic acids encoding proMMP polypeptides that exhibit constitutive enzymatic activity and isolated proMMP polypeptides.

FIELD OF THE INVENTION

The present invention pertains to transgenic mammals that expressrecombinant matrix-degrading enzymes in a temporally and spatiallyregulated manner. The invention further pertains to model systemsincorporating such transgenic mammals for studying degenerative jointdiseases, including systems for identifying therapeutic agents andtreatment regimens.

BACKGROUND OF THE INVENTION

Degenerative diseases of cartilage, including joint and disc diseasessuch as osteoarthritis, rheumatoid arthritis, andosteochondrodysplasias, are widespread, particularly in the elderly.Early symptoms common to these diseases include progressive loss ofproteoglycans in the joint (as evidenced by loss of metachromasia);collagen degradation; fibrillation of the cartilage surface; and,ultimately, loss of cartilage (which is evidenced radiologically asjoint space narrowing).

One of the primary targets affected by these diseases is type IIcollagen, the major structural collagen found in articular cartilage.There is a balance between the production of type II collagen andcatabolic enzymes that degrade type II collagen during normal remodelingof cartilage and bone. Pathological conditions such as, e.g.,degenerative joint diseases, may result when this balance is disrupted.

Among the enzymes that degrade extracellular matrix components arematrix metalloproteinases (MMPs), a family of zinc-dependent enzymes,and aggrecanase (Table 1).

TABLE 1 Matrix-Degrading Enzymes SUBSTRATES Collagen GelatinProteoglycan Fibronectin Laminin Elastin Other I. MetalloproteinasesCollagenases MMP-1 I, II, III, ✓ (intestinal collagenase) VII, X MMP-8I, II, III (neutrophil collagenase) MMP-13 I, II, III ✓ (collagenase 3)Gelatinases MMP-2 IV, V, ✓ ✓ ✓ ✓ (gelatinase A) VII, XI MMP-9 IV, V ✓ ✓(gelatinase B) Stromelysins MMP-3 ✓ ✓ ✓ ✓ activates (stromelysin 1) MMPzymogens MMP-7 IV ✓ ✓ ✓ ✓ ✓ (matrilysin) MMP-10 IV, V, ✓ ✓ ✓ activates(stromelysin 2) IX MMP zymogens MMP-11 IV ✓ ✓ activates serpins(stromelysin 3) Other MMP-12 ✓ (metalloelastase) MMP-14 ✓ proMMP-2,proMMP-13 MMP-15 MMP-16 proMMP-2 MMP-17 II. Aggrecanase  

MMPs are synthesized in articulating joints by chondrocytes, which, inmature articular cartilage, are terminally differentiated cells thatmaintain the cartilage-specific matrix phenotype. Overexpression of MMPsrelative to endogenous MMP inhibitors, as occurs in degenerative jointdiseases, may result in cartilage degradation. For example, Type IIcollagen is a substrate for MMP-13 and MMP-1 (Knauper et al., J. Biol.Chem. 271:1544, 1996) and both MMP-1 and MMP-13 proteins can be detectedimmunohistochemically in human osteoarthritic tissues. In some cases,MMP-13 and its cleavage products are found at higher levels than MMP-1.Billinghurst et al., J. Clin. Inves. 99:1534, 1997. Thus, MMP-13 mayplay an important role in cartilage degradation associated withosteoarthritis and other degenerative joint diseases. (Mitchell et al.,J. Clin. Inves. 97:761, 1996).

Animal models for osteoarthritis-related syndromes have been describedin guinea pigs (Watson et al., Arth. Rheum. 39: 1327, 1996) and in theinbred STR/ORT strain of mice (Das-Gupta et al., Int. J. Exp. Path.74:627, 1993). In guinea pigs, spontaneous osteoarthritis has a longcourse of development (six months or more), and only certain sublines ofSTR/ORT mice consistently develop degenerative joint disease. Thus, theduration and/or variability of these models renders them less applicableto drug discovery studies.

Other osteoarthritis-related models include surgically-induced jointdestabilization, e.g., anterior cruciate ligament transection and/orpartial meniscectomy in rabbits and dogs, which stimulates cartilagedegradation. Hulth et al., Acta Orthop. Scand. 41:522, 1970. Anothermodel employs injection of bacterial collagenase into the joints of ananimal to induce a biochemical ligament transection. Van der Kraan etal., J. Exp. Pathol. 71:19, 1990. Because (i) surgical or othermanipulation of individual animals is required; (ii) the animals arelarge and expensive; and/or (iii) the course of disease is notconsistent, these models cannot easily be used in large-scale studies,including drug screening.

Transgenic animal models, in principle, can provide the opportunity fora reproducible animal model system for degenerative joint diseases.However, previous attempts to engineer transgenic animals expressingMMPs such as MMP-1 and stromelysin have not resulted in an observablejoint degeneration phenotype in the transgenic animals. This could bedue to embryonic lethality caused by constitutive expression of theseenzymes. Witty et al., Mol.Biol. Cell 6:1287, 1995, have createdtransgenic animals that constitutively express MMP-1 and stromelysin inmammary tissue, but these animals do not exhibit symptoms ofosteoarthritis. D'Armiento et al., Cell 71:955, 1992, disclosetransgenic mice that express human interstitial collagenase in the lung.Liu et al., J. Cell Biol. 130:227, 1995, disclose transgenic animalsthat overexpress mutated type II collagen, resulting in connectivetissue defects but not osteoarthritis. None of these transgenic animalsystems provides a useful animal model for osteoarthritis. Khokha etal., Cancer and Metastasis Rev. 14:97, 1995; Shapiro, Matrix Biol.15:527, 1997.

Thus, there is a need in the art for animal model systems that mimichuman degenerative joint diseases such as, e.g., osteoarthritis,rheumatoid arthritis, and chondrodysplasias. Transgenic animalscontaining regulatable heterologous genes whose expression results incartilage degeneration are particularly advantageous in providingreproducible experimental control over the timing and the level ofexpression of the transgenes and, thereby, over the pathologicalsyndrome itself. Such animals can be used to determine what level ofexpression of the transgene is required to cause disease and,importantly, can be used for drug discovery and optimization oftreatment regimens. In particular, such transgenic animals can be usedto further define the role of matrix-degrading enzymes in cartilagedegradation and as an in vivo screen to identify compounds that modulatethese enzymes or compounds that inhibit the progression of degenerativejoint diseases.

SUMMARY OF THE INVENTION

The present invention provides transgenic non-human animals or theprogeny thereof whose somatic and germline cells contain, in stablyintegrated form, one or more heterologous or recombinant genes encodingpolypeptides comprising enzymatically active matrix-degrading enzymes(MDEs), preferably MMPs. MMPs for use in the invention comprise one ormore of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11,MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, and MMP-17; preferably one ormore of MMP-1, MMP-3, MMP-8, and MMP-13; and most preferably one or moreof MMP-1 and MMP-13; and include enzymatically active variants,fragments, and combinations of these polypeptides. Othermatrix-degrading enzymes can also be used, including, e.g., aggrecanase.The MDEs may be derived from any species, preferably human. In preferredembodiments, the recombinant MDE-encoding genes are selectivelyexpressed in articular chondrocytes of the transgenic animal andexpression results in pathological symptoms characteristic ofdegenerative joint disease.

In one aspect, the invention provides a transgenic animal or the progenythereof whose somatic and germline cells contain a stably integratedfirst recombinant gene encoding an MDE or an enzymatically activederivative or variant thereof, preferably a constitutively activeproMMP-13 variant (designated MMP-13*) comprising the sequence depictedin SEQ ID NO: 1. Preferably, the first recombinant gene is under thecontrol of a first regulatable promoter; most preferably, the firstregulatable promoter comprises a tet07 sequence, such as, e.g., thepromoter depicted in SEQ ID NO: 2. The transgenic animal may furthercomprise a second recombinant gene encoding a polypeptide that regulatesthe first regulatable promoter and is preferably a tTA polypeptide. Inthese embodiments, the second recombinant gene is under the control of asecond regulatable promoter, preferably one that comprises sequencesderived from a joint-specific promoter, and most preferably a type IIcollagen promoter, such as, e.g., the promoter depicted in SEQ ID NO: 3.Selective expression of the second recombinant gene in joint tissuesthus results in regulated joint-specific expression of the recombinantMDE.

In another aspect, the invention provides isolated nucleic acidsencoding enzymatically active MMP variants, preferably human proMMP-13variants, and most preferably MMP-13*. The invention also encompassesrecombinant cloning vectors comprising these nucleic acids; cellscomprising the vectors; methods for producing MMP-13-derivedpolypeptides comprising culturing the cells under conditions appropriatefor MMP-13 expression; and isolated MMP-13-derived polypeptides.

In yet another aspect, the invention provides methods for producingphenotypic changes characteristic of cartilage-degenerative disease in amammal, which comprise exposing the transgenic animals of the inventionto conditions that result in expression of the MDEs encoded by thetransgenes. In a preferred embodiment, a transgenic animal comprising afirst recombinant gene encoding MMP-13* operably linked to a tet07promoter and a second recombinant gene encoding a tTA protein operablylinked to a type II collagen promoter is maintained in the presence oftetracycline or a tetracycline analogue. When it is desired to induceexpression of MMP-13*, tetracycline or the tetracycline analogue iswithdrawn, MMP-13* is selectively expressed in joint tissues, andphenotypic changes characteristic of cartilage-degenerative diseaseresult.

In yet another aspect, the invention provides methods for determiningthe potential of a composition to counteract cartilage-degenerativedisease. The methods are carried out by administering a known dose ofthe composition to the transgenic animals of the invention, eitherbefore or after phenotypic indicators of cartilage-degenerative diseasehave developed; monitoring the indicators for a predetermined timefollowing administration of the composition; and comparing the extent ofthe indicators in the animal to which the composition was administeredrelative to a control transgenic animal that had not been exposed to thecomposition. Any difference in (i) the nature or extent of phenotypicindicators of cartilage-degenerative disease, (ii) the time required forthe indicators to develop, or (iii) the need for other ameliorativetreatments indicates the potential of the composition to counteractcartilage-degenerative disease.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent and Trademark Officeupon request and payment of the necessary fee.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent and Trademark Officeupon request and payment of the necessary fee.

FIG. 1A is a schematic illustration of the structure of human MMP-13(collagenase-3). The black box at the extreme aminoterminus representsthe pre domain (signal peptide) that targets nascent proMMP-13 forsecretion. The lightly hatched box represents the pro domain, SEQ ID NO:19, which is involved in maintaining the latency of the enzyme. Aconserved sequence within the pro domain that is important formaintaining enzyme latency is shown. The heavily hatched box representsthe 170-amino acid catalytic domain, SEQ ID NO: 20, which contains aconserved region (shown) that is important for catalytic activity. Theshaded box represents the 200-amino acid carboxyterminal domain.

FIG. 1B is an illustration of the nucleic acid sequence encoding aconstitutively active variant of human pro MMP-13, designated MMP-13*,SEQ ID NO: 18, and the amino acid sequence of MMP-13*, SEQ ID NO:1. Theresidues that are mutated relative to wild-type MMP-13, which aredepicted in larger type, are GTC at nucleotide positions 299-301.

FIGS. 2A and 2B are schematic illustrations of transgenes used forregulated expression of human MMP-13 * in transgenic mice. FIG. 2A showsa nucleic acid construct comprising, in a 5′ to 3′ direction: (i)sequences derived from rat type II collagen promoter; (ii) sequencesencoding a tetracycline repressor polypeptide fused in frame tosequences encoding a VP16 transcriptional activator polypeptide; and(iii) sequences comprising an SV40-derived RNA splice site andpolyadenylation signal. FIG. 2B shows a nucleic acid constructcomprising, in a 5′ to 3′ direction: (i) sequences derived from abacterial tet07 promoter; (ii) sequences encoding human MMP-13*; and(iii) sequences comprising an SV40-derived RNA splice site andpolyadenylation signal.

FIG. 3A is a schematic illustration of a transgene used to assesstissue-specific regulation conferred by a type II collagen promoter. Thenucleic acid construct comprises, in a 5′ to 3′ direction: (i) sequencesderived from a rat type II collagen promoter; (ii) sequences encodingbacterial β-galactosidase (LacZ); and (iii) sequences comprising anSV40-derived RNA splice site and polyadenylation signal.

FIG. 3B is a color photographic illustration of whole mount staining forβ-galactosidase activity of embryonic day 16 transgenic mouse embryosexpressing the transgene illustrated in FIG. 3A. Blue staining,indicating the presence of enzymatically active β-galactosidasepolypeptides, is evident in joints throughout the body of the transgenicanimal, while no staining is observed in the non-transgenic, wild-typelittermate.

FIG. 4 shows color photographic illustrations of immunohistochemicallocalization of type II collagen cleavage products in the growth plateand articular cartilage of transgenic mice expressing the transgenesshown in FIG. 2. The tissues were stained with an antibody thatrecognizes cleavage products of type II collagen. The left panel showstissue derived from a mouse that had been maintained on doxycycline torepress MMP-13* expression. The right panel shows tissue derived from amouse that had been withdrawn from doxycycline, allowing expression ofMMP-13*, for 30 days at 3 months of age.

FIG. 5 is a color photographic illustration of Safranin O staining ofthe articular cartilage and growth plate of the patella of doubletransgenic mice. The left panel shows tissue derived from a mousemaintained on doxycycline. The middle panel shows tissue derived from amouse 7 days after withdrawal from doxycycline. The right panel showstissue derived from a mouse 14 days after withdrawal from doxycycline.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that regulated expression ofmatrix-degrading enzymes in cartilage in transgenic mice results incharacteristic phenotypic changes associated with matrix degenerativediseases of the joints and intervertebral discs. The animal models ofthe invention provide novel model systems for matrix degenerativedisease syndromes which can be used for detailed characterization ofhuman joint and intervertebral disc pathologies as well as for drugdiscovery and optimization of treatment regimens.

A transgenic animal according to the invention is an animal having cellsthat contain a transgene which was introduced into the animal or anancestor of the animal at a prenatal (embryonic) stage. A transgenicanimal can be created, for example, by introducing the gene of interestinto the male pronucleus of a fertilized oocyte by, e.g.,microinjection, and allowing the oocyte to develop in a pseudopregnantfemale foster animal. The gene of interest may include appropriatepromoter sequences, as well as intronic sequences and polyadenylationsignal sequences. Methods for producing transgenic animals are disclosedin, e.g., U.S. Pat. Nos. 4,736,866 and 4,870,009 and Hogan et al., ALaboratory Manual, Cold Spring Harbor Laboratory, 1986. A transgenicfounder animal can be used to breed additional animals carrying thetransgene. A transgenic animal carrying one transgene can also be bredto another transgenic animal carrying a second transgene to create a“double transgenic” animal carrying two transgenes. Alternatively, twotransgenes can be co-microinjected to produce a double transgenicanimal. Animals carrying more than two transgenes are also possible.Furthermore, heterozygous transgenic animals, i.e., animals carrying onecopy of a transgene, can be bred to a second animal heterozygous for thesame transgene to produce homozygous animals carrying two copies of thetransgene.

The present invention encompasses transgenic animals, preferablymammals, which express MDEs, particularly MMPs, and most particularlythose MMPs having collagenase activity, from a recombinant gene. MDEsfor use in the invention include without limitation MMPs andaggrecanase. Useful MMPs include without limitation the collagenasesdesignated MMP-1, MMP-8 and MMP-13; the stromelysins designated MMP-3,MMP-10, and MMP-11; the gelatinases designated MMP-2 and MMP-9; themetalloelastase designated MMP-12; and membrane-type MMPs designatedMMP-14, MMP-15, MMP-16, and MMP-17. Matrisian, BioEssays, 14:455, 1992.Matrix-degrading activity as used herein refers to the proteolyticdegradation of matrix components, including, e.g., collagen,particularly type II collagen and most particularly the triple helicalform of type II collagen. Any polypeptide exhibiting matrix-degradingactivity may be used in practicing the invention, includingenzymatically active fragments of the above-described enzymes.Preferably, MMP-13 enzymatic activity is expressed. MMP-13 enzymaticactivity as used herein refers to the proteolytic degradation of type IIcollagen. Any MMP-13 polypeptide or fragment or derivative thereof thatexhibits MMP-13 enzymatic activity may be used. The enzymes may bederived from any animal species, including without limitation human,mouse, rat, rabbit, pig, cow, or non-human primate, or combinationsthereof. Preferably, the MMP-13 or derivative thereof is of humanorigin.

Normally, MMPs are synthesized as precursors (i.e., zymogens orproenzymes) whose enzymatic activity is latent; proteolytic removal ofthe pro region after secretion produces the enzymatically activeprotein. In preferred embodiments of the invention, the need forproteolytic processing is circumvented by the use of enzyme or proenzymevariants that are enzymatically active even when uncleaved. Suchvariants can be produced using conventional techniques for site-directedor random mutagenesis coupled with analysis of collagenase enzymaticactivity (see below). In this manner, modifications (including, e.g.,insertions, deletions, and substitutions), may be introduced into aproenzyme sequence, particularly within the pro region or near the proregion cleavage site, to produce a constitutively active polypeptidewhich does not require proteolytic processing for activation.Alternatively, the pro region may be deleted entirely. Furthermore,recombinant genes may be used in which the sequence encoding the nativesignal peptide is replaced by a heterologous sequence that functions asa signal peptide, i.e., promotes secretion. The use of genes encodingany such modified MMP polypeptides is encompassed by the invention.

Preferably, a constitutively active MMP-13 variant is used in practicingthe invention. Most preferably, the MMP-13 variant comprises a sequencecontaining a mutation in the sequence encoding the PRCGVPDV region, SEQID NO:4, specifically a substitution of Pro⁹⁹ to Val; the sequence ofthis polypeptide is depicted in SEQ ID NO: 1 and this polypeptide isdesignated MMP-13*. In another embodiment, the constitutively activeMMP-13 variant comprises a substitution of Val⁹⁸, SEQ ID NO: 21) to Gly.

The transgenic animals of the invention preferably express MMP activityin a regulated manner. Regulated expression as used herein refers totemporal and/or spatial control. Temporal control refers to the abilityto repress expression of MMP activity until a predetermined time in thedevelopment of the transgenic animal, after which MMP expression may beactivated and maintained for as long as desired. Preferably, MMPexpression is repressed throughout embryonic development and activatedin the adult animal. Spatial control refers to the ability toselectively express MMP activity in particular tissues. Preferably, MMPactivity is selectively expressed in joint tissues, most preferably inarticular chondrocytes.

Temporal control of MMP expression is achieved by use of one or morepolypeptides comprising a transcriptional repressor, a transcriptionalactivator or enhancer, or combinations thereof, in conjunction with apromoter responsive to the transcriptional repressor/activator used towhich the MMP-encoding sequence is operably linked. In one set ofembodiments, temporal control of MMP expression is achieved by (i)expression in the transgenic animal of a repressor polypeptide operablylinked to a polypeptide that directly or indirectly activatestranscription in eucaryotic cells, creating a repressor-activator fusionpolypeptide; and (ii) the coupled use of a target promoter operablylinked to an MMP-encoding sequence whose transcriptional activity isresponsive to the repressor-activator fusion polypeptide. Typically,nucleotide sequences encoding the repressor polypeptide are ligatedin-frame to sequences encoding the transcriptional activator polypeptideto create a chimeric gene encoding a fusion protein.

Useful repressor polypeptides include without limitation polypeptidescomprising sequences derived from bacterial repressors, includingwithout limitation tetracycline repressor, LacR repressor, KRAB domain,and lambda repressor (cro and cI), as well as eukaryotic repressors,including without limitation those involved in amino acid or sugarsynthesis. Useful direct transcriptional activator polypeptides includewithout limitation herpes simplex virus protein 16 (VP16); yeast GAL14;yeast STAT; steroid receptors such as, e.g., progesterone receptor andestrogen receptor; and constitutive activators such as, e.g., c-fos,c-jun, and SP-1. Alternatively, the repressor polypeptide may be linkedto a polypeptide that indirectly activates transcription by recruiting atranscriptional activator to interact with the repressor-activatorfusion protein; such indirect activator polypeptides include withoutlimitation TATA Box Binding Protein (TBP) and basic transcriptionfactors, including, e.g., basic transcription factor D.

According to the invention, each repressor-activator fusion protein isused in conjunction with a target promoter that is responsive to theparticular fusion protein and that regulates transcription of anMDE-encoding sequence. Typically, the promoter comprises at least oneoperator sequence responsive to the repressor component of therepressor-activator fusion polypeptide, which is operably linked to atleast a minimal promoter that supports transcription in eucaryoticcells. Examples of suitable repressor-responsive operator sequencesinclude without limitation sequences derived from the tetracyclineresistance operon encoded in Tn10 in E. coli, the lambda repressoroperon, and the yeast GAL repressor operon. Examples of suitableeucaryotic promoters from which minimal promoters may be derived includewithout limitation the cytomegalovirus (CMV) IE promoter, PtK-1(thymidine kinase) promoter, HSP (heat shock protein) promoter, and anyeukaryotic promoter containing a TATA box. Minimal promoter sequencesmay be derived from these promoters by (i) creating deletion mutantsusing conventional methods and (ii) testing the ability of the resultingsequences to activate transcription in a cell line. U.S. Pat. No.5,650,298 discloses a repressor-activator fusion protein comprised ofsequences derived from the tetracycline repressor fused to VP16sequences, which is designated tTA, and a tTA-responsive promoter,designated tet07, which comprises a Tn10-derived sequence linked to aportion of the CMV IE promoter.

Alternatively, temporal control is achieved by (i) expression in thetransgenic animal of a heterologous or recombinant transcriptionalactivator polypeptide or polypeptides and (ii) the coupled use of atarget promoter operably linked to an MMP-encoding sequence whosetranscriptional activity is responsive to the heterologous orrecombinant transcriptional activator. Useful transcriptional activatorsinclude without limitation a modified ecdysone receptor, in which a VP16transactivation domain linked to the aminoterminal transactivationdomain of the glucocorticoid receptor is fused to the ligand-bindingdomain and carboxyterminal sequence of the ecdysone receptor (No et al.,Proc. Natl. Acad. Sci. USA 93:3346, 1996); a chimeric protein,designated pGL-VP, comprising VP16 activator sequences, GAL4 activationsequences, and a mutated human progesterone receptor ligand-bindingdomain (Wang et al., Proc. Natl. Acad. Sci. USA 91:8180, 1994; Wang etal., Gene Therapy 4:432, 1997); and chimeric proteins comprisingtranscriptional activators fused to estrogen (or other steroid) bindingdomains (Mattioni et al., Meth. Cell Biol. 43:335, 1994). The ecdysonereceptor system utilizes retinoid X receptor (RXR) to form heterodimerswith the chimeric receptor, and responds to ecdysone, muristerone (anecdysone analogue) or dexamethasone. The pGL-VP system is responsive tomifepristone (RU486). Chimeric receptors containing an estrogen bindingdomain respond to hydroxytamoxifen (an estrogen analogue).

Spatial control of MDE expression is achieved by the use oftranscriptional promoters that direct transcription selectively in jointtissues. Joint-specific expression as used herein refers to expressionthat is greater in joints than in other cells; typically, the level ofexpression in non-joint tissues is less than 10% of the level ofexpression in joints. Preferably, expression in non-joint tissues isundetectable. Useful promoter sequences that confer joint-specificexpression on a sequence to which they are operably linked includewithout limitation sequences derived from the collagen type II promoter.It will be understood that a joint-specific promoter according to theinvention may comprise one or more copies of particular sequences orsub-sequences, and these sequences may be in direct or invertedorientation relative to each other and relative to the sequence whoseexpression is regulated by the promoter.

Coordinated spatial and temporal control of MDE expression is preferablyachieved by (i) placing expression of the repressor-activator fusionpolypeptide or the transcriptional activator polypeptide under thecontrol of a joint-specific promoter; (ii) placing the expression of theMDE or a derivative thereof under the control of a promoter responsiveto the repressor-activator fusion polypeptide or the transcriptionalactivator polypeptide; and (iii) maintaining the transgenic animalduring fetal development and early life under conditions in which MDEexpression is repressed.

The method by which transgenic animals are maintained during fetal andearly post-natal development so that MDE expression is repressed willdepend on the particular transgenes being expressed. When arepressor-activator fusion polypeptide is used, repression is achievedby providing the animal with an agent that binds to therepressor-activator fusion protein and results in repression oftranscription of the target MDE gene. In animals comprising a transgeneencoding a repressor-activator fusion polypeptide containing tetrepressor sequences, repression is achieved by providing tetracycline ora tetracycline analogue in the food or drinking water of the mother and,following birth, of the progeny. Tetracycline or an analogue may also beprovided using surgically implanted subcutaneous time-release pellets(Innovative Research of America, Inc., Sarasota Fla.) In this case,binding of tetracycline or a tetracycline analogue to therepressor-activator fusion protein prevents the fusion protein frombinding to, and activating transcription of, the cognate promoter.Tetracycline analogues are compounds closely related to tetracyclinewhich bind to the tet repressor with a Ka of at least about 10⁶M⁻¹,preferably with an affinity of about 10⁹M⁻¹ or greater. Usefultetracycline analogues include without limitation doxycycline,anhdryrotetracycline, chlortetracycline, epioxytetracycline, and thelike. The dosage used is one that will result in substantial repressionof MMP expression. Typically, tetracycline or a tetracycline analogue isadministered in the animal's drinking water at a dosage of about 1mg/ml. When it is desired that MMPs be expressed, the tetracycline oranalogue thereof is withheld.

In other embodiments, repression is achieved by withholding from theanimal an agent required for activity of the transcriptional activatorpolypeptide. For example, if the transcriptional activator is a modifiedecdysone receptor, the animals are maintained in the absence of ecdysoneor an ecdysone analogue throughout fetal and early post-nataldevelopment. Ecdysone analogues are compounds closely related toecdysone which bind to the modified ecdysone receptor with a Ka of atleast about 10⁶M⁻¹. Useful ecdysone analogues include without limitationmuristerone A. When it is desired that MDEs be expressed, the animalsare given, e.g., ecdysone or muristerone A via intraperitonealinjections at dosages of between about 10 mg and about 20 mg/animal.Similarly, when pGL-VP is used, activation is achieved by providingmifepristone.

In a preferred embodiment of the invention, a transgenic animal isconstructed whose somatic and germline cells contain in stablyintegrated form two recombinant genes: (i) a first recombinant genecomprising a sequence encoding MMP-13*, wherein the sequence is operablylinked to a tet07 promoter; and (ii) a second recombinant gene encodinga tTA protein operatively linked to a collagen type II promoter. In thisembodiment, animals are maintained in the presence of tetracycline or atetracycline analogue throughout fetal and early post-natal developmentto repress the gene. Afterwards, tetracycline or the tetracyclineanalogue is withdrawn, and MMP-13 enzymatic activity is selectivelyexpressed in joint tissues.

Animal Models for Cartilage-Degenerative Diseases

The present invention provides animal model systems in which phenotypicchanges characteristic of cartilage-degenerative diseases, such as,e.g., joint or disc disease, are reproducibly exhibited. These diseasesinclude without limitation osteoarthritis, rheumatoid arthritis,chondrodysplasias, and degenerative intervertebral disc diseases. Themodel systems of the invention exhibit one or more phenotypic indicatorscommon to these diseases, which include without limitation loss ofproteoglycan (as indicated by, e.g., loss of Safranin O staining) andcleavage of type II collagen in the affected tissues. The systemsencompass the transgenic animals described above, in which recombinantor heterologous MDEs, particularly MMPs, are expressed in cartilage at apredetermined time in the life of the transgenic animal. The timing ofthe appearance of cartilage-degenerative indicators is determined byactivating MDE expression and monitoring the effects on cartilage (seebelow). Preferably, one or more MDEs are expressed after birth, mostpreferably after the animal has reached adulthood.

Expression of the transgenes is typically monitored by extracting mRNAfrom different tissues and subjecting the extracted mRNA to one or moreof the following: (i) reverse transcriptase-polymerase chain reaction(RT-PCR), using primers homologous to the transgene; (ii) RNAaseprotection; and (iii) Northern blot analysis. Alternatively, in situhybridization may used.

The physiological effects of MDE expression on articular cartilage aremonitored in test animals by sacrificing the animals and subjectingparaffin-embedded decalcified cartilage to staining with (i) hematoxylinand eosin (using conventional techniques) followed by double stainingwith (ii) Safranin O and fast green. Peter et al., J. Exp. Pathol.71:19, 1990. Alternatively, frozen sections may be obtained and stainedwith antibodies that are specific for cleavage fragments derived fromtype II collagen. Billinghurst et al., J. Clin. Invest. 99:1534, 1997.Typically, expression of the MMP transgene(s) for at least about 7 daysresults in detectable loss of proteoglycan and changes in growth platemorphology (see, e.g., Example 5 below). Animal models in whichexpression of MDEs, particularly MMPs, and most particularly anenzymatically active form of MMP-13, results in proteoglycan loss and/orcleavage of type II collagen are within the scope of the invention.

Other phenotypic indicators of cartilage-degenerative disease which canbe monitored in transgenic animals produced according to the inventioninclude without limitation gross observations of changes in jointfunction and histological evidence of (i) fibrillation and loss ofarticular cartilage and (ii) osteophyte formation.

Syndromes for which the transgenic animals of the invention provideuseful models include without limitation any pathological condition thatmanifests a disturbance in the composition, morphology, and/or functionof cartilage, including osteoarthritis; rheumatoid arthritis;degenerative intervertebral disc diseases; chondrodysplasias, including,e.g., Kniest dysplasia, achondrogenesis, and hypophosphatasia; andproteoglycan-mediated disorders, such as occur, e.g., in brachymorphicanimals. Hall et al., Cartilage: Molecular Aspects, CRC Press, 1991, pp.201-203.

In further embodiments of the invention, the transgenic animals can besubjected to additional treatments to modulate thecartilage-degenerative indicators and/or to supplement the animals'disease phenotype with additional physiological effects such as, e.g.,those associated with a particular disease. For example, the transgenicanimals may be further treated with inflammatory mediators to augmentcollagen degradation and/or induce loss of proteoglycan (see, e.g.,Example 6 below). Furthermore, the timing and extent of MDE induction,with or without additional treatments, can be adapted to replicate thesymptomatology of a particular disease or syndrome.

Methods for Evaluating Drugs that Modulate Degenerative Diseases ofCartilage

The present invention encompasses methods for discovery and evaluationof drugs and therapies for their efficacy against degenerative diseasesof cartilage, particularly degenerative joint diseases. In oneembodiment of the invention, the transgenic animals of the invention aremaintained under conditions in which expression of one or more MDEsresults in one or more phenotypic indicators of cartilage-degenerativedisease. Once the symptoms have developed, the potential of acomposition to counteract cartilage-degenerative disease can beevaluated by administering a known dose of the composition to the animalin which the symptoms have developed; monitoring the phenotypicindicators for a predetermined time following administration of thecomposition; and comparing the extent of the phenotypic indicators inthe animal to which the composition was administered relative to acontrol animal. Control animals comprise age- and sex-matched transgenicanimals that are maintained under an identical regimen (i.e., expressthe transgenes) but which do not receive the composition. Anystatistically significant difference in the extent or nature of thephenotypic indicators indicates the potential of the composition tocounteract cartilage-degenerative disease. As used herein, phenotypicindicators of cartilage-degenerative disease refer to proteoglycan loss,joint space narrowing, collagen degradation, and destruction ofcartilage.

In another embodiment of the invention, the potential of a compositionto counteract degenerative diseases of cartilage, particularlydegenerative joint disease, is evaluated by administering to atransgenic animal a known dose of the composition before and/orsimultaneous with the induction of MDE expression in the transgenicanimal; monitoring phenotypic indicators of cartilage-degenerativedisease for a predetermined time following administration of thecomposition and MDE induction; and comparing the extent of thephenotypic indicators and/or disease in the animal to which thecomposition was administered relative to a control animal that had notbeen exposed to the composition. In this embodiment, any statisticallysignificant difference in the extent or nature of the phenotypicindicators and/or disease, or any statistically significant delay inappearance of the phentoypic indicators or disease, indicates thepotential of the composition to counteract cartilage-degenerativedisease.

A further indication of the potential of a composition to counteractcartilage-degenerative disease is the ability of the composition tocause any reduction in the extent or duration of other treatments,including, e.g., the dosage and timing of administration of othertherapeutic agents used to alleviate symptoms of the disease.

Compounds that may be tested for anti-cartilage-degenerative diseasepotential may be found in, for example, natural product libraries,fermentation libraries (encompassing plants and microorganisms),combinatorial libraries, compound files, synthetic compound libraries,and compounds resulting from directed rational drug design andsynthesis. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), andMicrosource (New Milford, Conn.). A rare chemical library is availablefrom Aldrich Chemical Company, Inc. (Milwaukee, Wis.). Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available from, for example, Pan Laboratories(Bothell, Wash.) or MycoSearch (N.C.), or are readily producible.Additionally, natural and synthetically produced libraries and compoundsare readily modified through conventional chemical, physical, andbiochemical means. Blondelle et al., TibTech 14:60, 1996.

Transgenic animals

Transgenic animals as used herein refers to animals into which one ormore heterologous and/or recombinant genes have been introduced. Thetransgenes may be from a different species, or from the same species asthe transgenic animal but are not naturally found in the animal in theconfiguration and/or at the chromosomal locus conferred by thetransgene. Transgenes may comprise foreign DNA sequences, i.e.,sequences not normally found in the genome of the host animal.Alternatively or additionally, transgenes may comprise endogenous DNAsequences that have been rearranged or mutated in vitro in order toalter the normal in vivo pattern of expression of the gene, or to alteror eliminate the biological activity of an endogenous gene productencoded by the gene. Also encompassed by the invention are DNA fragmentsthat are introduced into a pre-existing gene to, e.g., change patternsof expression or to provide additional means of regulating theexpression of the gene. Watson et al., “The Introduction of ForeignGenes Into Mice,” in Recombinant DNA, 2d Ed., W. H. Freeman & Co., NewYork, 1992, pp. 255-272; Gordon, J. W., Intl. Rev. Cytol. 115:171,1989;Jaenisch, Science 240:1468, 1989; Rossant, Neuron 2:323, 1990.

The transgenic non-human animals of the invention are produced byintroducing transgenes into the germline of the non-human animal.Embryonal target cells at various developmental stages are used tointroduce the transgenes of the invention. Different methods are useddepending on the stage of development of the embryonal target cell(s).Such methods include, but are not limited to, microinjection of zygotes,viral integration, and transformation of embryonic stem cells asdescribed below.

1. Microinjection of zygotes is the preferred method for incorporatingtransgenes into animal genomes. A zygote, which is a fertilized ovumthat has not undergone pronuclei fusion or subsequent cell division, isthe preferred target cell for microinjection of transgenic DNAsequences. The murine male pronucleus reaches a size of approximately 20micrometers in diameter, a feature which allows for the reproducibleinjection of 1-2 picoliters of a solution containing transgenic DNAsequences. The use of a zygote for introduction of transgenes has theadvantage that, in most cases, the injected transgenic DNA sequenceswill be incorporated into the host animal's genome before the first celldivision. Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438, 1985. Asa consequence, all cells of the resultant transgenic animals (founderanimals) stably carry an incorporated transgene at a particular geneticlocus, referred to as a transgenic allele. The transgenic alleledemonstrates Mendelian inheritance, i.e., half of the offspringresulting from the cross of a transgenic animal with a non-transgenicanimal will inherit the transgenic allele, in accordance with Mendel'srules of random assortment.

2. Viral integration can also be used to introduce the transgenes of theinvention into an animal. The developing embryos are cultured in vitroto the blastocyte developmental stage. The blastomeres may be infectedwith appropriate retroviruses. Jaenich, Proc. Natl. Acad. Sci. USA73:1260. Infection of the blastomeres is enhanced by enzymatic removalof the zona pellucida. Transgenes are introduced via viral vectors whichare typically replication-defective but which remain competent forintegration of viral-associated DNA sequences, including transgenic DNAsequences linked to such viral sequences, into the host animal's genome.Transfection is easily and efficiently obtained by culture ofblastomeres on a monolayer of cells producing the transgene-containingviral vector. Alternatively, infection may be performed using cells at alater developmental stage, such as blastocoeles. In any event, mosttransgenic founder animals produced by viral integration will be mosaicsfor the transgenic allele; that is, the transgene is incorporated intoonly a subset of all the cells that form the transgenic founder animals.Moreover, multiple viral integration events may occur in a singlefounder animal, generating multiple transgenic alleles which willsegregate in future generations of offspring. Introduction of transgenesinto germline cells by this method is possible but probably occurs at alow frequency. However, once a transgene has been introduced intogermline cells by this method, offspring may be produced in which thetransgenic allele is present in all of the animal's cells, i.e., in bothsomatic and germline cells.

3. Embryonal stem (ES) cells can also serve as target cells forintroduction of the transgenes of the invention into animals. ES cellsare obtained from pre-implantation embryos that are cultured in vitro.Evans et al., Nature 292:154, 1981. ES cells that have been transformedwith a transgene can be combined with an animal blastocyst, after whichthe ES cells colonize the embryo and contribute to the germline of theresulting animal (which is a chimera, i.e., composed of cells derivedfrom two or more animals). Again, once a transgene has been introducedinto germline cells by this method, offspring may be produced in whichthe transgenic allele is present in all of the animal's cells, i.e., inboth somatic and germline cells.

Although the initial introduction of a transgene is a Lamarckian(non-Mendelian) event, the transgenes of the invention may be stablyintegrated into germ line cells and transmitted to offspring of thetransgenic animal as Mendelian loci. Other transgenic techniques resultin mosaic transgenic animals, in which some cells carry the transgenesand other cells do not. In mosaic transgenic animals in which germ linecells do not carry the transgenes, transmission of the transgenes tooffspring does not occur. Nevertheless, mosaic transgenic animals arecapable of demonstrating phenotypes associated with the transgenes.

In practicing the invention, animals of the transgenic maintenance lineare crossed with animals having a genetic background in which expressionof the transgene results in symptoms of cartilage-degenerative disease.Offspring that have inherited the transgenes of the invention aredistinguished from littermates that have not inherited transgenes byanalysis of genetic material from the offspring for the presence ofnucleic acid sequences derived from the transgenes of the invention. Forexample, biological fluids that contain polypeptides uniquely encoded bythe transgenes of the invention may be immunoassayed for the presence ofthe polypeptides. A simpler and more reliable means of identifyingtransgenic offspring comprises obtaining a tissue sample from anextremity of an animal, such as, for example, a tail, and analyzing thesample for the presence of nucleic acid sequences corresponding to theDNA sequence of a unique portion or portions of the transgenes of theinvention. The presence of such nucleic acid sequences may be determinedby, e.g., hybridization (“Southern”) analysis with DNA sequencescorresponding to unique portions of the transgene, analysis of theproducts of PCR reactions using DNA sequences in a sample as substrates,oligonucleotides derived from the transgene's DNA sequence, and thelike.

Nucleic Acids, Vectors, Expression Systems, and Polypeptides

The present invention encompasses isolated nucleic acids encoding MDEs,particularly MMPs, and enzymatically active fragments derived therefrom,as well as constitutively active MMP variants and enzymatically activefragments derived therefrom. The invention also encompasses complementsof the above nucleic acids; vectors comprising the nucleic acids; cellscomprising the vectors; and isolated polypeptides encoded by the nucleicacids.

Many techniques in molecular biology, microbiology, recombinant DNA, andprotein biochemistry are used in practicing the present invention, suchas those explained in, for example, Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A PracticalApproach, Volumes I and II, 1985 (D. N. Glover ed.); OligonucleotideSynthesis, 1984, (M. L. Gait ed.); Transcription and Translation, 1984(Hames and Higgins eds.); A Practical Guide to Molecular Cloning; theseries, Methods in Enzymology (Academic Press, Inc.); and ProteinPurification: Principles and Practice, Second Edition (Springer-Verlag,N.Y.).

“Nucleic acid” or “polynucleotide” as used herein refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides.This includes single- and double-stranded molecules, such as, forexample, DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “proteinnucleic acids” (PNA) formed by conjugating bases to an amino acidbackbone. This also includes nucleic acids containing modified bases.

A “coding sequence” or a “protein-coding sequence” is a polynucleotidesequence capable of being transcribed into mRNA and/or capable of beingtranslated into a polypeptide. The boundaries of the coding sequence aretypically determined by a translation start codon at the 5′-terminus anda translation stop codon at the 3′-terminus.

A “complement” of a nucleic acid sequence as used herein refers to the“antisense” sequence that participates in Watson-Crick base-pairing withthe original sequence.

An “isolated” nucleic acid or polypeptide as used herein refers to acomponent that is removed from its original environment (for example,its natural environment if it is naturally occurring). An isolatednucleic acid or polypeptide typically contains less than about 50%,preferably less than about 75%, and most preferably less than about 90%,of the cellular components with which it was originally associated.

A nucleic acid or polypeptide sequence that is “derived from” adesignated sequence refers to a sequence that corresponds to a region ofthe designated sequence. For nucleic acid sequences, this encompassessequences that are homologous or complementary to the sequence, as wellas “sequence-conservative variants” and “function-conservativevariants.” For polypeptide sequences, this encompasses“function-conservative variants.” Sequence-conservative variants arethose in which a change of one or more nucleotides in a given codonposition results in no alteration in the amino acid encoded at thatposition. Function-conservative variants are those in which a givenamino acid residue in a polypeptide has been changed without alteringthe overall conformation and function of the native polypeptide,including, but not limited to, replacement of an amino acid with onehaving similar physico-chemical properties (such as, for example,acidic, basic, hydrophobic, and the like). “Function-conservative”variants also include any polypeptides that have the ability to elicitantibodies specific to a designated polypeptide.

Nucleic acids comprising any of the sequences disclosed herein orsubsequences thereof can be prepared by conventional methods. Forexample, DNA can be chemically synthesized using, e.g., thephosphoramidite solid support method of Matteucci et al., 1981, J. Am.Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem.764:17078, or other well known methods. This can be performed bysequentially linking a series of oligonucleotide cassettes comprisingpairs of synthetic oligonucleotides.

Due to the degeneracy of the genetic code, many different nucleotidesequences can encode polypeptides having the amino acid sequencesdefined herein or subsequences thereof. The codons can be selected foroptimal expression in prokaryotic or eukaryotic systems. Such degeneratevariants are also encompassed by this invention.

The nucleic acids may also be modified by many means known in the art.Non-limiting examples of such modifications include methylation, “caps”,substitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as, for example, thosewith uncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Nucleic acids may containone or more additional covalently linked moieties, such as, for example,proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. PNAs are also encompassed by the term “nucleicacid”. The nucleic acid may be derivatized by formation of a methyl orethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore,the nucleic acid sequences of the present invention may also be modifiedwith a label capable of providing a detectable signal, either directlyor indirectly. Exemplary labels include radioisotopes, fluorescentmolecules, biotin, and the like.

The polypeptides of the invention may be expressed by using many knownvectors, such as pUC plasmids, pET plasmids (Novagen, Inc., Madison,Wis.), or pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), andmany appropriate host cells, using methods disclosed or cited herein orotherwise known to those skilled in the relevant art. The particularchoice of vector/host is not critical to the practice of the invention.Recombinant cloning vectors will often include one or more replicationsystems for cloning or expression; one or more markers for selection inthe host, such as, for example, antibiotic resistance; and one or moreexpression cassettes. The inserted coding sequences may be synthesizedby standard methods, isolated from natural sources, prepared as hybrids,or the like. Ligation of the coding sequences to transcriptionalregulatory elements and/or to other amino acid coding sequences may beachieved by known methods. Suitable host cells may betransformed/transfected/infected as appropriate by any suitable methodincluding electroporation, CaCl₂ mediated DNA uptake, fungal infection,microinjection, microprojectile, or other established methods.

Appropriate host cells include bacteria, archebacteria, fungi, yeast,plant, and animal cells, and especially mammalian cells. Of particularinterest are E. coli, S. aureus, B. subtilis, Saccharomyces cerevisiae,Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129cells, 293 cells, Neurospora, CHO cells, COS cells, HeLa cells, andimmortalized mammalian myeloid and lymphoid cell lines. Preferredreplication systems include M13, ColE1, SV40, baculovirus, lambda,adenovirus, cytomegalovirus, and the like. A large number oftranscription initiation and termination regulatory regions have beenisolated and are effective in the transcription and translation ofheterologous proteins in the various hosts. Examples of these regions,methods of isolation, manner of manipulation, etc. are known in the art(Ausubel et al., Current Protocols in Molecular Biology, John Wiley,1997). Under appropriate expression conditions, host cells can be usedas a source of recombinantly produced peptides and polypeptides.

The MDEs of the present invention, including function-conservativevariants, may be isolated from native or heterologous organisms or cells(including, but not limited to, bacteria, fungi, insect, plant, andmammalian cells) into which the protein-coding sequence has beenintroduced and expressed. Alternatively, these polypeptides may beproduced in cell-free protein synthesis systems, which may additionallybe supplemented with microsomal membranes to achieve glycosylation andsignal peptide processing of preprocollagenases. Furthermore, thepolypeptides may be chemically synthesized by commercially availableautomated procedures, including, without limitation, exclusive solidphase synthesis, partial solid phase methods, fragment condensation, orclassical solution synthesis.

Methods for polypeptide purification are well-known in the art,including, without limitation, preparative disc-gel electrophoresis,isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ionexchange and partition chromatography, and countercurrent distribution.For some purposes, it is preferable to produce the polypeptide in arecombinant system in which the protein contains an additional sequencetag that facilitates purification, such as, but not limited to, apolyhistidine sequence. The polypeptide can then be purified from acrude lysate of the host cell by chromatography on an appropriatesolid-phase matrix. Alternatively, antibodies produced against theprotein or against peptides derived therefrom can be used aspurification reagents. Other purification methods are possible.

The construction and analysis of MMP variants and derivatives thatexhibit enzymatic activity, and preferably constitutive enzymaticactivity, can be achieved by routine application of conventionalmethods. First, a nucleic acid encoding an MMP is modified either bysite-directed or random mutagenesis, or is used in a construction schemeas one segment of a fusion gene. Preferably, the procedure results in amodification either contained within the sequence encoding the proregion or near the pro region cleavage site; this includes deleting thepro region entirely. Alternatively, sequences may be constructed thatencode fusion proteins either between enzymatically active MMP domainsand other polypeptides, or between different MMPs. The modified nucleicacid is then used to program synthesis of a variant MMP, either in acell-free system, in intact cells (including permeabilized cells), or ina transgenic animal. Preferably, either a cell-free system or a cellculture system is used to express the MMP variant or derivative. Theextent of pro region cleavage is assessed by metabolic labelling andresolution of the MMP product by SDS-PAGE. Finally, MMP enzymaticactivity is measured using conventional assays, such as, by quantifyingthe cleavage of natural substrates or model peptides, as disclosed,e.g., in Weingarten et al., Biochem. 24:6730, 1985; Woessner et al., J.Biol. Chem., 263:16918, 1988, and Knight et al., FEBS Letts., 296:263,1992. In this manner, a large number of MMP variants and derivatives,including, e.g., function-conservative variants of MMP-13*, can becreated routinely and assayed for MMP enzymatic activity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples are intended to illustrate the present inventionwithout limitation.

EXAMPLE 1 Construction of a Gene Encoding a Modified, ConstitutivelyActive ProMMP-13

The following experiments were performed to create a gene encoding aprocollagenase derived from MMP-13 that is enzymatically active in theabsence of pro region cleavage. The sequence of this proMMP-13 variant,designated MMP13*, is shown in FIG. 1B, SEQ ID NO: 1.

Site-directed mutagenesis was performed to modify MMP-13 cDNA asfollows:

A cDNA fragment encoding proMMP was obtained by digesting plasmid pNot3A(Freije et al., J. Biol. Chem. 269:16766, 1994; GENBANK accession numberX75308) with Xbal and HindIII and purifying the resulting ˜1515 bpfragment. This fragment was subcloned into theTet-resistant/Amp-sensitive palter plasmid (Promega, Madison, Wis.) thathad been digested with XbaI and HindIII.

Site-directed mutagenesis was performed using the Altered Sites II invitro Mutagenesis System (Promega, Madison, Wis.). Briefly, phagemidsingle-stranded DNA was purified from cultures containing the helperphage R408 (Promega). In addition to the Amp repair—Tet knock-outconversion oligos (Promega), an oligonucleotide having the sequence5′-AAGCCAAGATGCGGGGTTGTCGATGTGGGTGAATACAAT-3′, SEQ ID NO:5, wasphosphorylated and annealed to the single-stranded DNA, followed bymutant strand synthesis. The reaction mixture was then used to transformthe repair-minus E. coli strain ES1301 mutS, and the culture was grownin ampicillin selective media. Plasmid DNA was isolated from isolatedclones and transformed into JM109 cells, which were then plated on LBplates containing 120 μg/ml ampicillin.

The above procedure resulted in a proline-to-valine substitution atamino acid 99. The modified proMMP was designated MMP13* (FIG. 1B, SEQID NO:1).

Using a similar technique, site-directed mutagenesis was also used tointroduce a valine to glycine mutation at amino acid 98. A mutagenicoligonucleotide having the sequence5′-GAAAAAGCCAAGATGCGGGGGTCCTGATGTGGGTGAATAC-5′, SEQ ID NO:6 was used asdescribed above. This procedure resulted in a valine-to-glycinesubstitution at amino acid 98.

After confirmation of the above mutations by direct sequencing, cDNAencoding MMP13* cDNA was excised from the pAlter vector by digestionwith Eco RI and Hind III.

The enzymatic activity of MMP-13* was determined as follows:

1. cDNAs encoding both mutant forms of MMP13 and wild-type MMP-13 weresubcloned into a BS(SK−) vector (Stratagene) containing the CMV promoter(Xho I-Eco RI) and the SV40 splice poly (A)n (Xba I-Nco I). Duplicatecultures of Hela cells (10 cm dishes) were transfected with 50 μg ofthese plasmids using the CaPO₄ precipitation method (Promega). Fivehours later, cells were subjected to a 1-minute glycerol shock using asolution containing an equal volume of 2×HBS+30 % glycerol. Thisprocedure is described in the Profection Mammalian Transfection Systemstechnical manual (Promega).

2. Twenty-four hours following transfection, the culture medium (D-MEMcontaining 10% fetal bovine serum) was replaced with D-MEM containing noserum and 10 μM CGS-27023A (Ciba-Geigy), an MMP inhibitor. It isbelieved that, in the absence of an added MMP inhibitor, MMPs producedby the culture autodigest; thus, addition of an MMP inhibitor to theculture medium resulted in a detectable MMP13 band.

3. Forty-eight hours after the addition of serum-free medium containingthe MMP inhibitor, 10 ml of supernatant were collected and concentratedabout 200-fold using Centriprep-30 and Centricon-10 concentrators(Amicon), after which an equal volume of 2× Tris-glycine SDS runningbuffer was added to each sample. The samples were then applied to a4-16% pre-stained beta-casein zymogram SDS polyacrylamide gel (Novex).After electrophoresis, the gels were renatured in renaturing buffer(Novex) for 30 minutes at room temperature, followed by overnightincubation at 37° C. in zymogram developing buffer (Novex).

The results indicated that cells expressing either a variant MMP13containing a proline->valine substitution at position 99 (MMP-13*) or avariant MMP13 containing a valine->glycine substitution at position 98secreted detectable MMP activity similar to cells expressing wild-typeMMP-13. This method thus provides a rapid screen for MMP13 variants thatretain MMP13 enzymatic activity.

In a further step, cDNA encoding MMP-13* was operably linked to atranscriptional regulatory sequence derived from the tet07 promoter asfollows:

1. The BS(SK−) vector (Stratagene) was digested with KpnI and NotI. Asynthetic duplex oligonucleotide having the following sequence wasdigested with KpnI and Not I and ligated to the vector:

5′-GGTACCACTAGTAAGCTTAGATCTCATATGGTCGACCCCGGGGAATTCCTGCAGGGATCCTCTAGAAGTACTCCATGGGTATACATCGATGCGGCCGC-3′, SEQ ID NO:7

The SB(SK−) vector as modified above was digested with XbaI and NcoI. A745 bp fragment containing the SV40 splice site and polyadenylationsignal, which was obtained by digesting pcDNAI/Amp (Invitrogen,Carlsbad, Calif.) with XbaI and Ncol, was ligated to this vector. This745 bp fragment was recovered by digestion of the vector with XbaI andNotI and was inserted into the original BS(SK−) vector.

2. The resulting vector was linearized by digestion with XhoI and EcoRIand ligated to a 460 bp XhoI-EcoRI fragment containing the tet07promoter region (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547,1992). This vector was then digested with SpeI, blunt-ended with Klenowpolymerase, and digested with EcoRI.

3. pAlter-MMP13* was digested with HindIII, blunt-ended with Klenowpolymerase, and digested with EcoRI to obtain an MMP13*-encodingfragment.

4. The MMP13* EcoRI fragment was cloned into the EcoRI digested vectorobtained in step 2.

5. The 2792 bp transgene, SEQ ID NO:8, was excised by digestion withXhoI and NotI and purified using CsCl gradient centrifugation.

EXAMPLE 2 Construction of a Collagen Type II-Promoter-Linked tTa gene

The following experiments were performed to create a gene encoding a tTArepressor-activator fusion protein operably linked to a joint-specific(type II collagen) promoter as well as a reporter gene suitable forassessing the tissue-specific expression conferred by the type IIcollagen promoter.

1. Type II collagen promoter- tetracycline/VP16 transgene: The modifiedBS(SK−) vector containing the SV40 splice site and polyadenylationsignal as described in Example 1 above was digested with NdeI and Sma Iand ligated to a 1897 bp fragment containing the collagen II promoterand enhancer. This fragment was obtained by digesting plasmid PBSΔH1with HindIII, after which it was blunt-ended with Klenow and digestedwith NdeI.

The plasmid was then digested with EcoRI and BamHI and ligated to a 1025bp fragment encoding the tetracycline/VP16 repressor-activator fusionprotein that had been excised from the pUHG15-1 plasmid (Gossen et al.,Proc. Natl. Acad. Sci. USA 89:5547, 1992) using EcoRI and BamHI. Theplasmid was linearized by digestion with BglII, dephosphorylated usingcalf intestinal phosphatase, and ligated to a 1554 BamHI enhancerfragment obtained from plasmid PBSΔH1.

Finally, the 5276 bp transgene, SEQ ID NO:9, was excised from the vectorby digestion with Kpnl and NotI, gel purified, purified by CsCl gradientcentrifugation, dialyzed against microinjection buffer (5 mM Tris-HCl pH7.4, 0.1 mM EDTA pH 8.0) and used for microinjection (see Example 3below). 2. Type II collagen promoter-β-galactosidase gene: A 4179 bpBamHI-BglII fragment containing the β-galactosidase gene fused to theβ-globin splice sequence and polyadenylation signal was excised fromplasmid pUGH16-3 (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547,1992) and cloned into the BamHI site of unmodified BS(SK−) (Stratagene).This plasmid was digested with EcoRI and HindIII and ligated to a 655 bpHind III-Eco RI fragment containing the type II collagen promotersequence, which was excised from the plasmid described in (1) above. Theplasmid was then digested with EcoRI and ligated to a 2807 bp Eco RIfragment which had been excised from the type II collagen promoterplasmid described above. Restriction mapping was used to verify theorientation of each insert. The 7664 bp transgene, SEQ ID NO:10, wasexcised by digestion with HindIII and NotI, gel purified, purified byCsCl gradient centrifugation, dialyzed against microinjection buffer (5mM Tris pH 7.4, 0.1 mM EDTA pH 8.0), and microinjected into mouseembryos.

EXAMPLE 3 Production and Characterization of Transgenic Mice ExpressingTetracycline-Regulated MMP-13 in Joint Tissues

The following experiments were performed to produce transgenic miceexpressing MMP-13* or a LacZ (β-galactosidase) reporter gene.

To produce mice expressing MMP-13* under tetracycline regulation, anXhoI-NotI tet07-MMP-13* DNA fragment (FIG. 2B, SEQ ID NO:8) and aKpnI-NotI CPE-tTA DNA fragment (FIG. 2A, SEQ ID NO:9) wereco-microinjected into fertilized mouse embryos in equimolar amounts. Toproduce mice expressing the reporter gene, a HindIII-NotI LacZ fragment(described in Example 2 above) was injected into (FVB/N) fertilized eggsas described (Hogan et al., Manipulating the Mouse Embryo, Cold SpringHarbor Laboratories, 1996).

Founder animals were first identified by PCR as follows. ThetTA-encoding transgene was identified using a primer corresponding tothe tTA sequence (5′-CGAGGGCCTGCTCGATCTCC-3′, SEQ ID NO:11) and a primercorresponding to a 3′ untranslated sequence (5′-GGCATTCCACCACTGCTCCC-3′,SEQ ID NO:12). The resulting PCR product was 584 bp in size. TheMMP13*-encoding transgene was identified using primers corresponding tosequences encoding MMP13* (5′-GAGCACCCTTCTCATGACCTC-3′, SEQ ID NO:13)and the 3′ untranslated region, respectively. The resulting PCR productwas 731 bp in size. Out of 112 newborn mice, 7 transgenic foundersharboring both transgenes were found.

The LacZ-encoding transgene was identified using primers correspondingto the nuclear localization signal of the β-galactosidase gene(5′-GTTGGTGTAGATGGGCGCATCG-3′, SEQ ID NO:14) and the collagen promoter(5′-GCGGGGTCTCAGGTTACAGCC-3′, SEQ ID NO: 15). The resulting PCR productwas 673 bp in size.

Southern blot analysis of tail DNA digested with BamHI/NcoI orPvuII/NcoI and hybridized to the 3′ untranslated region under highstringency conditions was performed to confirm the results obtainedusing PCR. The number of copies of transgene DNA that integrated intothe genome was determined by comparing the relative intensity of thehybridization signal from transgenic mice with that obtained usingcontrol DNAs containing 10 and 100 genome equivalents of the same DNAthat was injected. Transgenic lines were generated by mating founderanimals to FVB/N wild type animals.

All mice were administered doxycycline (Sigma Chemical Co., St. LouisMo.) at a concentration of 1.0 mg/ml in acidic drinking water, which waschanged on a daily basis. Schultze et al., Nature Biotech. 14:499, 1996.

EXAMPLE 4 Analysis of Joint-Specific Expression Conferred by Type IICollagen Promoter Constructs

The following experiments were performed to evaluate tissue-specificexpression conferred by use of the type II collagen promoter.

Joint-specific expression was monitored by staining for β-galactosidaseactivity as described (Hogan et al., Manipulating the Mouse Embryo, ColdSpring Harbor Laboratories, 1996); using this method, the presence ofenzymatically active β-galactosidase is reflected in the appearance of ablue stain.

Wild-type female mice were mated with transgenic males harboring theCPE-LacZ construct as described in Examples 2 and 3 above. On embryonicday 16, the females were sacrificed, and the embryos were stained forβ-galactosidase activity. Prior to fixation, the tails were removed fromthe embryos and used as a source of template DNA for PCR reactions todetermine transgene transmission.

FIG. 3B illustrates the blue staining that is observed in jointsthroughout the body of the embryonic day 16 transgenic mouse embryo.Specifically, β-galactosidase expression was observed in ankles, knees,hips, phalanges, wrists, elbows, shoulders, and vertebrae. In additionto the cartilage of the joints, cartilage that had not ossified to boneat this stage of development, i.e., some of the facial, skull, and ribbones, also expressed β-galactosidase. No staining was observed innon-transgenic, wild type littermates.

These results indicated that the type II collagen-derived promoteraccording to the present invention is capable of conferringjoint-specific expression on sequences to which it is operably linked.

EXAMPLE 5 Analysis of the Phenotypic Effects of Joint-SpecificExpression of MMP-13*

The following experiments were performed to evaluate the development ofphenotypic indicators of cartilage degeneration in transgenic animalsexpressing MMP-13* in joint tissue.

Mice harboring both the tet07-MMP-13* and CPE-tTA constructs (producedas described in Example 3 above) were maintained on doxycyline untiladulthood (approximately 8 weeks postpartum).

Expression of MMP13* was first evaluated in hemizygous mice using RT-PCRto detect the transcripts. No expression of the transgene was observedin any of the lines in any of the tissues sampled, including brain,heart, liver, kidney, hindlimb, muscle, bone, or tongue.

To examine whether MMP-13* DNA in any of the double-transgenic lines wascapable of being expressed, embryonic fibroblasts were prepared fromthese animals and transfected with a tTA expression plasmid (Gossen andBujard, Proc.Natl.Acad. Sci. USA 89:5547, 1992). The transfection wasdone because the joint-specific type II collagen promoter regulating tTAexpression (and thereby MMP-13* expression) in the transgenic animalsmight not be expected to function in embryonic fibroblasts.

For this purpose, wild-type females were mated to transgenic malesharboring both the type II collagen promoter-linked tTA andTet07-MMP-13* transgenes. On embryonic day 15, the females weresacrificed, and fibroblasts were prepared from the embryos (Graham etal., Virol. 52:456, 1973; Lopata et al., Nuc. Acid Res. 12:5707, 1984).The fibroblasts were cultured in DMEM containing 10% fetal bovine serum,and were transfected with the tTa expression plasmid using the calciumphosphate method. Forty-eight hours after transfection, total RNA wasprepared from the cells using the Trizol method (GIBCO/BRL, GrandIsland, N.Y.). RT-PCR was performed using the Superscriptpreamplification system (GIBCO/BRL) for first-strand cDNA, after whichMMP-13* sequences were detected by PCR using the followingMMP-13*-specific primers: 5′-GCCCTCTGGCCTGCTGGCTCATG-3′, SEQ ID NO:16and 5′-CAGGAGAGTCTTGCCTGTATCCTC-3′, SEQ ID NO:17.

Fibroblasts from several transgenic lines (such as, e.g., lines 8 and42) were capable of expressing MMP13*, as evidenced by the appearance ofa PCR product of the predicted size. No MMP13* RT-PCR band was detectedfrom cells transfected with vehicle alone. These results indicated that,in these mice, the MMP13* transgene is integrated into atranscriptionally active region of the chromatin.

Expression of MMP13* in the double transgenic lines was further analyzedby immunohistochemistry, using antibodies specific for MMP-13-derivedtype II collagen cleavage fragments. For this purpose, joints were fixedin 4% paraformaldehyde in PBS at neutral pH for 60 minutes at roomtemperature. They were then rinsed twice in PBS, incubated in 0.1MTris-HCl, pH 7.4, overnight, and partially decalcified in 0.2M EDTA atneutral pH. The samples were transferred to TOC medium and 6-mm frozensections were obtained using a Hacker/Bright cryostat. The sections werestained with an antibody that recognizes an epitope present in adegradation product of type II collagen, specifically, in the TC^(A)degradation product, which is also designated the ¾ piece. Billinghurstet al., J. Clin. Invest. 99:1534, 1997.

As early as 3 days after removal of the mice from doxycycline, MMP-13cleavage products could be detected. After 30 days without doxycycline,a substantial increase in staining in the growth plate and in thearticular cartilage could be seen (FIG. 4), but the results differedamong different lines of mice (see Table 1 below).

TABLE 1 Immunohistochemistry Loss of F1 Days hMMP13 Type II CollagenSafranin Animal off Dox Ab Cleavage Fragments Ab O Stain Line 8 wt − −not remarkable Line 8  0 d − − ″  3 d + + ″  7 d ++ ++ Mild 14 d +++ +++Moderate wt − − not remarkable Line 6 30 d Moderate Line 8 30 d +++ +++Moderate Line 42 30 d + − not remarkable

To study the effect of MMP13* activity on cartilage in adult transgenicanimals, mice were withdrawn from doxycycline for increasing times,after which they were sacrificed. Paraffin-embedded formaldehyde-fixedsections of decalcified cartilage were sectioned and stained with (i)hematoxylin and eosin and (ii) Safranin O followed by fast green(American Histo Labs, Gaithersburg Md.). Peter et al., J. Exp. Pathol.71:19, 1990.

Control transgenic animals that lack MMP13* expression retain asignificant amount of safranin O stain in both the articular cartilageas well as the growth plate of their patella (FIG. 5, left panel). Bycontrast, transgenic animals from line 8 show a substantial loss ofsafranin O staining in their joints following doxycyline withdrawal.After seven days, a mild reduction of safranin O staining is observed inthe articular cartilage of the patella (FIG. 5, middle panel), whichprogresses by day 14 to moderate loss of stain in articular cartilage aswell as the growth plate (FIG. 5, right panel). A significant loss ofsafranin O stain was also observed in the other joints including thecartilage of the tarsus and femur, as well as wrist and knuckle,indicating a reduced proteoglycan concentration in these areas comparedto controls.

EXAMPLE 6 Augmentation of the Development of Symptoms of JointDegenerative Disease in MMP-13 Transgenic Mice

The following treatments are performed to enhance the symptoms of jointdegeneration exhibited by the transgenic animals of the invention.

A group of transgenic mice are treated to induce expression of thetransgenes at 4-12 weeks of age. Two to six weeks after induction, themice are injected intraperitonealy with an inflammatory agent, includingwithout limitation, lipopolysaccharide (10-100 μg), zymosan (1-10 mg),the superantigen Staphylococcal Enterotoxin B (1-100 μg), or TGF-β (1-10μg). Alternatively, the animals are injected intraarticularly with aninflammatory or chondrocyte function-modulating agent, including withoutlimitation, lipopolysaccharide (1-100 ng), zymosan (50-250 μg), papain(10-100 μg), TGF-β (0.01-1 μg), Bone Morphogenic Protein −2 (2-1000 ng),IL-1 (1-100 ng), TGF-α (10-200 ng), IGF (0.01-1 μg), or FGF (0.01-1 μg).Age- and sex-matched transgenic mice maintained under a regimen in whichthe transgenes are not expressed receive the same treatment and serve ascontrols.

The development of symptoms of degenerative joint disease is monitoredby gross observation of joint swelling and function, and by histologicalevaluation of the joint at selected timepoints after exposure to theinflammatory agent.

The agents will induce an acute inflammatory response and/or transientloss of proteoglycan with a duration of less than one week. The acuteinflammatory response and/or transient cartilage changes will upregulategene expression in the chondrocytes, enhancing the expression of thetransgene and increasing the levels of MMP-13 produced.

All patents, applications, articles, publications, and test methodsmentioned above are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description.Such obvious variations are within the full intended scope of theappended claims.

21 471 amino acids amino acid single linear protein 1 Met His Pro GlyVal Leu Ala Ala Phe Leu Phe Leu Ser Trp Thr His 1 5 10 15 Cys Arg AlaLeu Pro Leu Pro Ser Gly Gly Asp Glu Asp Asp Leu Ser 20 25 30 Glu Glu AspLeu Gln Phe Ala Glu Arg Tyr Leu Arg Ser Tyr Tyr His 35 40 45 Pro Thr AsnLeu Ala Gly Ile Leu Lys Glu Asn Ala Ala Ser Ser Met 50 55 60 Thr Glu ArgLeu Arg Glu Met Gln Ser Phe Phe Gly Leu Glu Val Thr 65 70 75 80 Gly LysLeu Asp Asp Asn Thr Leu Asp Val Met Lys Lys Pro Arg Cys 85 90 95 Gly ValVal Asp Val Gly Glu Tyr Asn Val Phe Pro Arg Thr Leu Lys 100 105 110 TrpSer Lys Met Asn Leu Thr Tyr Arg Ile Val Asn Tyr Thr Pro Asp 115 120 125Met Thr His Ser Glu Val Glu Lys Ala Phe Lys Lys Ala Phe Lys Val 130 135140 Trp Ser Asp Val Thr Pro Leu Asn Phe Thr Arg Leu His Asp Gly Ile 145150 155 160 Ala Asp Ile Met Ile Ser Phe Gly Ile Lys Glu His Gly Asp PheTyr 165 170 175 Pro Phe Asp Gly Pro Ser Gly Leu Leu Ala His Ala Phe ProPro Gly 180 185 190 Pro Asn Tyr Gly Gly Asp Ala His Phe Asp Asp Asp GluThr Trp Thr 195 200 205 Ser Ser Ser Lys Gly Tyr Asn Leu Phe Leu Val AlaAla His Glu Phe 210 215 220 Gly His Ser Leu Gly Leu Asp His Ser Lys AspPro Gly Ala Leu Met 225 230 235 240 Phe Pro Ile Tyr Thr Tyr Thr Gly LysSer His Phe Met Leu Pro Asp 245 250 255 Asp Asp Val Gln Gly Ile Gln SerLeu Tyr Gly Pro Gly Asp Glu Asp 260 265 270 Pro Asn Pro Lys His Pro LysThr Pro Asp Lys Cys Asp Pro Ser Leu 275 280 285 Ser Leu Asp Ala Ile ThrSer Leu Arg Gly Glu Thr Met Ile Phe Lys 290 295 300 Asp Arg Phe Phe TrpArg Leu His Pro Gln Gln Val Asp Ala Glu Leu 305 310 315 320 Phe Leu ThrLys Ser Phe Trp Pro Glu Leu Pro Asn Arg Ile Asp Ala 325 330 335 Ala TyrGlu His Pro Ser His Asp Leu Ile Phe Ile Phe Arg Gly Arg 340 345 350 LysPhe Trp Ala Leu Asn Gly Tyr Asp Ile Leu Glu Gly Tyr Pro Lys 355 360 365Lys Ile Ser Glu Leu Gly Leu Pro Lys Glu Val Lys Lys Ile Ser Ala 370 375380 Ala Val His Phe Glu Asp Thr Gly Lys Thr Leu Leu Phe Ser Gly Asn 385390 395 400 Gln Val Trp Arg Tyr Asp Asp Thr Asn His Ile Met Asp Lys AspTyr 405 410 415 Pro Arg Leu Ile Glu Glu Asp Phe Pro Gly Ile Gly Asp LysVal Asp 420 425 430 Ala Val Tyr Glu Lys Asn Gly Tyr Ile Tyr Phe Phe AsnGly Pro Ile 435 440 445 Gln Phe Glu Tyr Ser Ile Trp Ser Asn Arg Ile ValArg Val Met Pro 450 455 460 Ala Asn Ser Ile Leu Trp Cys 465 470 470 basepairs nucleic acid single linear 2 CTCGAGTTTA CCACTCCCTA TCAGTGATAGAGAAAAGTGA AAGTCGAGTT TACCACTCCC 60 TATCAGTGAT AGAGAAAAGT GAAAGTCGAGTTTACCACTC CCTATCAGTG ATAGAGAAAA 120 GTGAAAGTCG AGTTTACCAC TCCCTATCAGTGATAGAGAA AAGTGAAAGT CGAGTTTACC 180 ACTCCCTATC AGTGATAGAG AAAAGTGAAAGTCGAGTTTA CCACTCCCTA TCAGTGATAG 240 AGAAAAGTGA AAGTCGAGTT TACCACTCCCTATCAGTGAT AGAGAAAAGT GAAAGTCGAG 300 CTCGGTACCC GGGTCGAGTA GGCGTGTACGGTGGGAGGCC TATATAAGCA GAGCTCGTTT 360 AGTGAACCGT CAGATCGCCT GGAGACGCCATCCACGCTGT TTTGACCTCC ATAGAAGACA 420 CCGGGACCGA TCCAGCCTCC GCGGCCCCGAATTAGCTTGA TATCGAATTC 470 3479 base pairs nucleic acid single linear 3GGTACCACTA GTAAGCTTAG ATCCACTGTC TGGGATTATA TCAGGACAAC CGAAGCCTGG 60AAAGTGTATT AGGTAGAGCA TTTTCTTCCA CGTGTTTGGG CACGTTTCCG ACAGCTAGGA 120TTCCAGCTCT GTCTTTGTAT GTTACAGACT GTAAATCAAT CGCAGGTGAA ACTGTTTGGA 180CAGTAGGTGG GGATCAAAGA CCCTCCGCCC GTGAGACTCT AGGCGCTTTC CCCTGCCACC 240AGCCTGTCTC CAGAGATGCT CTGGAAGGAG GCGGGCCCGG GCGGTCTTTC TGCTCTTTAG 300CGTGGCGGAC GCGGCGGCGG GGGCAGGGCT GGAGCAGAGA GCGCTGCAGT GATAGAACTT 360TCTGACCCCG CTGCGCAGGG CGGCAGGGTG GCAGGGTGGC AGGGTGGCGA GCTAAGCCAG 420AGCCGAACGC TGGAGCTCTG GGAGGAACAT CGAAGGTTTG TATGTGGTCT GAGATCGGCC 480TGACTATATT TTTTTGTCCT AAATTTGCAA GCACACACCC ACAAAGCTGC GGTCTTGACC 540GGTATTCTTT ATAGAGCGCA ATGGAGTGAG CTGAGTGTCT AAACGATTTC CCTAATTCAT 600CTGATAGCAG AGGCGCTCTC CTAATTGGCG AAGAGCTGCC TCATGTCCGC AACTTTTTGG 660CAGAGTGAAT TCCACAGCTT TGTGTGTGTG TGTGGGGGGG GGTGTAAGGG GTGTCTAAAA 720CTTTCGGTCT CCTACTATTC TGTATCTCGA CCGGTTGGTT TTACACCCCG GCTCATCTCA 780TCAACGCAAA CACCCCCACT CTCCTATGGA CCCAAGGACC TGACGTGGGG GAAGGTGGAC 840ATTAGGAATG TCAGAAACCT AGAGTCCACG CTCCTCCTCT CCATCTTTCC ACGAGTTTGG 900GAAACTTCTT GGCTGCGAAG ACTTTGACCC ACATCTGCAT TTCTCAGCCC CAGCTTCCAA 960AAGTGCTGCA GGTTCGGGAG GGGAGACCTC AGTCCTCCTT TGTGAGGCTT GTTTGCGTTG 1020AGGGATTGGC AGCGATGGCT TCCAGATGGG CTGAAACCCT GCCCGTATTT ATTTAAACTG 1080GTTCCTCGTG GAGAGCTGTG AATCGGGCTC TGTATGCGCT CGAGAAAAGC CCCATTCATG 1140AGAGGCAAGG CCCAGTGGGT CCCCCCGACT CCCCGACCCC CCTCTCCCAC AATATATCCC 1200CCCTCCCTGT GCCCGCCTGC CGCCACCTCC CGGGCTCCGG CCCCGCGCGC AGCGGCGACG 1260AAGCAACACA GTTCCCCGAA AGAGGTAGCT TTTTAATTGG CCAGCCACAA AGAATCACTT 1320ATGCCGCACG GCGGTAACGA GGGGAACCGG ATCGGGCGGC CAGGATGCTA TCTGTGTAGC 1380CCTTTTCGTG CCACAATTAG GGTGGTGCTG GCTTCCTCCG ACCGCACCTA GGCGATCTGG 1440TTACACTGTT GGCTCCTTTC TTGGGCAGTC ATTTAATCCT ACTTTTTACT CTACGAATGT 1500CTGTCTGATG GAGGGCTGTG TCCGGAGCCC CATCCACAAA GAGTCAGCCA GCAGCTCTCA 1560CACCCGGCTG GATCTCATAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT 1620AAGCCAGCCA AGCTAGCTTG CGCAAGCTAG CTTGCGATCC GTAAAAATGT GTGAGAGTTA 1680CAAAATGTCT TCCGGGCTAA GATCCGACAG CCATGGTCCA AAGAAGACTT CGGCACTGCA 1740GACTTAAAAC CAGCTTTCTA GCAGAGGCAG AAGGATCTAG AGCCAAAGGC AAAGACTTGA 1800ATAGGCTGGG AAGATGCAAG AATGGCATTT TACATAAAGA ACACTCTCTC CTTTTCCAGC 1860CAGCACACTT GCATAGAAAT TAAGTTTTAC ACTTGAAGTT CTTTGTTTCC ATCCTGAGAA 1920GCTCCAAAGT CTGAGGTGGT GTGGTATGCT GGGTAATTCT CCCCACCCCC CAACATTCCC 1980TGGGGGTTCC ATGGGGGTAG CTTCTCCCAA GGACTTCCAG CGGCAACACA GAAATCCCAC 2040TTCGAGACAA AGGAGTTACT GCTTAAATCA GGCCCTAATT TCCAAGGTTC CCTTTGCTTA 2100AAGTTCCCTA GAGGACCATC TCACTTCTAA AGAAAAGGTG TATTCGGGGA CCCATCCTCA 2160ACCTCCTTGT TATGGAAGGA GACTTCGGGA ACAGAGCAAG GGCTGAGCCT CCGGCAGTTT 2220GGGGTAAGGT TGGGGTTGGG GGGAGCAAGG AAGGCAAGTG AGGCTGGAGG CCCAGGGATA 2280GGGGAAGATG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTC TCGGGGATGG 2340TGGTGGTGGA CAACTAGGAA ACTCTGGCGC TTTCTCCTCC CCTCACAAAA CTGAGTCCAG 2400CTGGAGCCGC CTCCAGACTC TCTGGCCAGG GCCTCAGAGT GGTCAACAGT CCCTGGCCAG 2460CGTTGCTCTC TCCAGGCTAA GGGCACCCAC TCCCCTGGAG ATTCCTGAAC CTGGGCCAGG 2520AAGAGCCGAA TTAGACAAGT GTCTCCAATC CGGCTGCGTG CGGATTTTGT TGCGGTGTCC 2580CTCGGTTGTC TGCAGTTCCT TTAGTCCCTT CCCTGGCCTG CCCCTTACAC CTCCACACAG 2640GTCCCCCTCT GTGTAGGAAT ACACCAGACC CTCTCTTAGC CACACACACC TCCAGTCCCC 2700CGTCTACCTA GATTTTTTTC ATAGCTAGTT GGATGGGGGA TGGGTTAGGG AGGCTGGGTT 2760TGCGAGCCTC CAGGTGGGAG TTCACCGACA GGTACTCCGC AAAGGAGCTG GAAGGCAGGT 2820CTGGAAAACT GTCCCCCAGA TTTAGGATTC TGGGCAGCTT CCATCAGCTT ATACTTTGGC 2880TCCCCCGCCC CCTAAACTCC CCATCCCCAC CTTCCTTTCT CCCGTTACTT CGTCCTCCCT 2940CGCCTTTCCA GCCTTGAGTC TAAAGCTCCA TGCTTATGCC TCTGCAAACA ACCCCCTCCC 3000TTCTAACCCC AGCAGAACTC CGAGGAAAGG GGCCGGAGGC CCCCCTTCTC GCCTGTGGTT 3060AGAGGGGGCA GTGTGGCAGT CCCAAGTGGG GGCGACCGGA GGCCGTCTCG GTGCCCCGCC 3120CGATCAGGCC ACTGGGCACA TCGGGGGCGG GAAGCTGGGC TCACCAAAGG GGCGACTGGC 3180CTTGGCAGGT GTGGGCTCTG GTCCGGCCTG GGCAGGCTCC GGGGGCGGGG TCTCAGGTTA 3240CAGCCCCGCG GGGGGCTGGG GGGCGGCCCG CGGTTTGGGC TGGTTTGCCA GCCTTTGGAG 3300CGACCGGGAG CATATAACCG GAGCCTCTGC TGGGAGAAGA CGCAGAGCGC CGCTGGGCTG 3360CCGGGTCTCC TGCCTCCTCC TCCTGCTCCT AGAGCCTCCT GCATGAGGGC GCGGTAGAGA 3420CCCGGACCCG CTCCGTGCTC TGCCGCCTCG CCGAGCTTCG CCCGCAAGCT GGGGAATTC 3479 8amino acids amino acid single linear peptide 4 Pro Arg Cys Gly Val ProAsp Val 1 5 39 base pairs nucleic acid single linear 5 AAGCCAAGATGCGGGGTTGT CGATGTGGGT GAATACAAT 39 40 base pairs nucleic acid singlelinear 6 GAAAAAGCCA AGATGCGGGG GTCCTGATGT GGGTGAATAC 40 98 base pairsnucleic acid single linear 7 GGTACCACTA GTAAGCTTAG ATCTCATATG GTCGACCCCGGGGAATTCCT GCAGGGATCC 60 TCTAGAAGTA CTCCATGGGT ATACATCGAT GCGGCCGC 982792 base pairs nucleic acid single linear cDNA 8 CTCGAGTTTA CCACTCCCTATCAGTGATAG AGAAAAGTGA AAGTCGAGTT TACCACTCCC 60 TATCAGTGAT AGAGAAAAGTGAAAGTCGAG TTTACCACTC CCTATCAGTG ATAGAGAAAA 120 GTGAAAGTCG AGTTTACCACTCCCTATCAG TGATAGAGAA AAGTGAAAGT CGAGTTTACC 180 ACTCCCTATC AGTGATAGAGAAAAGTGAAA GTCGAGTTTA CCACTCCCTA TCAGTGATAG 240 AGAAAAGTGA AAGTCGAGTTTACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 300 CTCGGTACCC GGGTCGAGTAGGCGTGTACG GTGGGAGGCC TATATAAGCA GAGCTCGTTT 360 AGTGAACCGT CAGATCGCCTGGAGACGCCA TCCACGCTGT TTTGACCTCC ATAGAAGACA 420 CCGGGACCGA TCCAGCCTCCGCGGCCCCGA ATTAGCTTGA TATCGAATTC GAGCTCGGTA 480 CCCGGGGATC CTCTAGACAAGATGCATCCA GGGGTCCTGG CTGCCTTCCT CTTCTTGAGC 540 TGGACTCATT GTCGGGCCCTGCCCCTTCCC AGTGGTGGTG ATGAAGATGA TTTGTCTGAG 600 GAAGACCTCC AGTTTGCAGAGCGCTACCTG AGATCATACT ACCATCCTAC AAATCTCGCG 660 GGAATCCTGA AGGAGAATGCAGCAAGCTCC ATGACTGAGA GGCTCCGAGA AATGCAGTCT 720 TTCTTCGGCT TAGAGGTGACTGGCAAACTT GACGATAACA CCTTAGATGT CATGAAAAAG 780 CCAAGATGCG GGGTTGTCGATGTGGGTGAA TACAATGTTT TCCCTCGAAC TCTTAAATGG 840 TCCAAAATGA ATTTAACCTACAGAATTGTG AATTACACCC CTGATATGAC TCATTCTGAA 900 GTCGAAAAGG CATTCAAAAAAGCCTTCAAA GTTTGGTCCG ATGTAACTCC TCTGAATTTT 960 ACCAGACTTC ACGATGGCATTGCTGACATC ATGATCTCTT TTGGAATTAA GGAGCATGGC 1020 GACTTCTACC CATTTGATGGGCCCTCTGGC CTGCTGGCTC ATGCTTTTCC TCCTGGGCCA 1080 AATTATGGAG GAGATGCCCATTTTGATGAT GATGAAACCT GGACAAGTAG TTCCAAAGGC 1140 TACAACTTGT TTCTTGTTGCTGCGCATGAG TTCGGCCACT CCTTAGGTCT TGACCACTCC 1200 AAGGACCCTG GAGCACTCATGTTTCCTATC TACACCTACA CCGGCAAAAG CCACTTTATG 1260 CTTCCTGATG ACGATGTACAAGGGATCCAG TCTCTCTATG GTCCAGGAGA TGAAGACCCC 1320 AACCCTAAAC ATCCAAAAACGCCAGACAAA TGTGACCCTT CCTTATCCCT TGATGCCATT 1380 ACCAGTCTCC GAGGAGAAACAATGATCTTT AAAGACAGAT TCTTCTGGCG CCTGCATCCT 1440 CAGCAGGTTG ATGCGGAGCTGTTTTTAACG AAATCATTTT GGCCAGAACT TCCCAACCGT 1500 ATTGATGCTG CATATGAGCACCCTTCTCAT GACCTCATCT TCATCTTCAG AGGTAGAAAA 1560 TTTTGGGCTC TTAATGGTTATGACATTCTG GAAGGTTATC CCAAAAAAAT ATCTGAACTG 1620 GGTCTTCCAA AAGAAGTTAAGAAGATAAGT GCAGCTGTTC ACTTTGAGGA TACAGGCAAG 1680 ACTCTCCTGT TCTCAGGAAACCAGGTCTGG AGATATGATG ATACTAACCA TATTATGGAT 1740 AAAGACTATC CGAGACTAATAGAAGAAGAC TTCCCAGGAA TTGGTGATAA AGTAGATGCT 1800 GTCTATGAGA AAAATGGTTATATCTATTTT TTCAACGGAC CCATACAGTT TGAATACAGC 1860 ATCTGGAGTA ACCGTATTGTTCGCGTCATG CCAGCAAATT CCATTTTGTG GTGTTAAGTG 1920 TCTTTTTAAA AATTGTTATTTAAATCCTGA AGAGCATTTG GGGTAATACT TCCAGAAGTG 1980 CGGGGTAGGG GAAGAAGAGCTATCAGGAGA AAGCTCTAGT TCTAGAGGGC CCTATTCTAT 2040 AGTGTCACCT AAATGCTAGAGGATCTTTGT GAAGGAACCT TACTTCTGTG GTGTGACATA 2100 ATTGGACAAA CTACCTACAGAGATTTAAAG CTCTAAGGTA AATATAAAAT TTTTAAGTGT 2160 ATAATGTGTT AAACTACTGATTCTAATTGT TTGTGTATTT TAGATTCCAA CCTATGGAAC 2220 TGATGAATGG GAGCAGTGGTGGAATGCCTT TAATGAGGAA AACCTGTTTT GCTCAGAAGA 2280 AATGCCATCT AGTGATGATGAGGCTACTGC TGACTCTCAA CATTCTACTC CTCCAAAAAA 2340 GAAGAGAAAG GTAGAAGACCCCAAGGACTT TCCTTCAGAA TTGCTAAGTT TTTTGAGTCA 2400 TGCTGTGTTT AGTAATAGAACTCTTGCTTG CTTTGCTATT TACACCACAA AGGAAAAAGC 2460 TGCACTGCTA TACAAGAAAATTATGGAAAA ATATTTGATG TATAGTGCCT TGACTAGAGA 2520 TCATAATCAG CCATACCACATTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC 2580 TCCCCCTGAA CCTGAAACATAAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG 2640 CTTATAATGG TTACAAATAAAGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT 2700 CACTGCATTC TAGTTGTGGTTTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGGATCA 2760 TCCCGCCATG GGTATACATCGATGCGGCCG CC 2792 5276 base pairs nucleic acid single linear cDNA 9GGTACCACTA GTAAGCTTAG ATCCACTGTC TGGGATTATA TCAGGACAAC CGAAGCCTGG 60AAAGTGTATT AGGTAGAGCA TTTTCTTCCA CGTGTTTGGG CACGTTTCCG ACAGCTAGGA 120TTCCAGCTCT GTCTTTGTAT GTTACAGACT GTAAATCAAT CGCAGGTGAA ACTGTTTGGA 180CAGTAGGTGG GGATCAAAGA CCCTCCGCCC GTGAGACTCT AGGCGCTTTC CCCTGCCACC 240AGCCTGTCTC CAGAGATGCT CTGGAAGGAG GCGGGCCCGG GCGGTCTTTC TGCTCTTTAG 300CGTGGCGGAC GCGGCGGCGG GGGCAGGGCT GGAGCAGAGA GCGCTGCAGT GATAGAACTT 360TCTGACCCCG CTGCGCAGGG CGGCAGGGTG GCAGGGTGGC AGGGTGGCGA GCTAAGCCAG 420AGCCGAACGC TGGAGCTCTG GGAGGAACAT CGAAGTGTTT GTATGTGGTC TGAGATCGGC 480CTGACTATAT TTTTTTGTCC TAAATTTGCA AGCACACACC CACAAAGCTG CGGTCTTGAC 540CGGTATTCTT TATAGAGCGC AATGGAGTGA GCTGAGTGTC TAAACGATTT CCCTAATTCA 600TCTGATAGCA GAGGCGCTCT CCTAATTGGC GAAGAGCTGC CTCATGTCCG CAACTTTTTG 660GCAGAGTGAA TTCCACAGCT TTGTGTGTGT GTGTGGGGGG GGGTGTAAGG GGTGTCTAAA 720ACTTTCGGTC TCCTACTATT CTGTATCTCG ACCGGTTGGT TTTACACCCC GGCTCATCTC 780ATCAACGCAA ACACCCCCAC TCTCCTATGG ACCCAAGGAC CTGACGTGGG GGAAGGTGGA 840CATTAGGAAT GTCAGAAACC TAGAGTCCAC GCTCCTCCTC TCCATCTTTC CACGAGTTTG 900GGAAACTTCT TGGCTGCGAA GACTTTGACC CACATCTGCA TTTCTCAGCC CCAGCTTCCA 960AAAGTGCTGC AGGTTCGGGA GGGGAGACCT CAGTCCTCCT TTGTGAGGCT TGTTTGCGTT 1020GAGGGATTGG CAGCGATGGC TTCCAGATGG GCTGAAACCC TGCCCGTATT TATTTAAACT 1080GGTTCCTCGT GGAGAGCTGT GAATCGGGCT CTGTATGCGC TCGAGAAAAG CCCCATTCAT 1140GAGAGGCAAG GCCCAGTGGG TCCCCCCGAC TCCCCGACCC CCCTCTCCCA CAATATATCC 1200CCCCTCCCTG TGCCCGCCTG CCGCCACCTC CCGGGCTCCG GCCCCGCGCG CAGCGGCGAC 1260GAAGCAACAC AGTTCCCCGA AAGAGGTAGC TTTTTAATTG GCCAGCCACA AAGAATCACT 1320TATGCCGCAC GGCGGTAACG AGGGGAACCG GATCGGGCGG CCAGGATGCT ATCTGTGTAG 1380CCCTTTTCGT GCCACAATTA GGGTGGTGCT GGCTTCCTCC GACCGCACCT AGGCGATCTG 1440GTTACACTGT TGGCTCCTTT CTTGGGCAGT CATTTAATCC TACTTTTTAC TCTACGAATG 1500TCTGTCTGAT GGAGGGCTGT GTCCGGAGCC CCATCCACAA AGAGTCAGCC AGCAGCTCTC 1560ACACCCGGCT GGATCTCATA TGGTGCACTC TCAGTACAAT CTGCTCTGAT GCCGCATAGT 1620TAAGCCAGCC AAGCTAGCTT GCGCAAGCTA GCTTGCGATC CGTAAAAATG TGTGAGAGTT 1680ACAAAATGTC TTCCGGGCTA AGATCCGACA GCCATGGTCC AAAGAAGACT TCGGCACTGC 1740AGACTTAAAA CCAGCTTTCT AGCAGAGGCA GAAGGATCTA GAGCCAAAGG CAAAGACTTG 1800AATAGGCTGG GAAGATGCAA GAATGGCATT TTACATAAAG AACACTCTCT CCTTTTCCAG 1860CCAGCACACT TGCATAGAAA TTAAGTTTTA CACTTGAAGT TCTTTGTTTC CATCCTGAGA 1920AGCTCCAAAG TCTGAGGTGG TGTGGTATGC TGGGTAATTC TCCCCACCCC CCAACATTCC 1980CTGGGGGTTC CATGGGGGTA GCTTCTCCCA AGGACTTCCA GCGGCAACAC AGAAATCCCA 2040CTTCGAGACA AAGGAGTTAC TGCTTAAATC AGGCCCTAAT TTCCAAGGTT CCCTTTGCTT 2100AAAGTTCCCT AGAGGACCAT CTCACTTCTA AAGAAAAGGT GTATTCGGGG ACCCATCCTC 2160AACCTCCTTG TTATGGAAGG AGACTTCGGG AACAGAGCAA GGGCTGAGCC TCCGGCAGTT 2220TGGGGTAAGG TTGGGGTTGG GGGGAGCAAG GAAGGCAAGT GAGGCTGGAG GCCCAGGGAT 2280AGGGGAAGAT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT CTCGGGGATG 2340GTGGTGGTGG ACAACTAGGA AACTCTGGCG CTTTCTCCTC CCCTCACAAA ACTGAGTCCA 2400GCTGGAGCCG CCTCCAGACT CTCTGGCCAG GGCCTCAGAG TGGTCAACAG TCCCTGGCCA 2460GCGTTGCTCT CTCCAGGCTA AGGGCACCCA CTCCCCTGGA GATTCCTGAA CCTGGGCCAG 2520GAAGAGCCGA ATTAGACAAG TGTCTCCAAT CCGGCTGCGT GCGGATTTTG TTGCGGTGTC 2580CCTCGGTTGT CTGCAGTTCC TTTAGTCCCT TCCCTGGCCT GCCCCTTACA CCTCCACACA 2640GGTCCCCCTC TGTGTAGGAA TACACCAGAC CCTCTCTTAG CCACACACAC CTCCAGTCCC 2700CCGTCTACCT AGATTTTTTT CATAGCTAGT TGGATGGGGG ATGGGTTAGG GAGGCTGGGT 2760TTGCGAGCCT CCAGGTGGGA GTTCACCGAC AGGTACTCCG CAAAGGAGCT GGAAGGCAGG 2820TCTGGAAAAC TGTCCCCCAG ATTTAGGATT CTGGGCAGCT TCCATCAGCT TATACTTTGG 2880CTCCCCCGCC CCCTAAACTC CCCATCCCCA CCTTCCTTTC TCCCGTTACT TCGTCCTCCC 2940TCGCCTTTCC AGCCTTGAGT CTAAAGCTCC ATGCTTATGC CTCTGCAAAC AACCCCCTCC 3000CTTCTAACCC CAGCAGAACT CCGAGGAAAG GGGCCGGAGG CCCCCCTTCT CGCCTGTGGT 3060TAGAGGGGGC AGTGTGGCAG TCCCAAGTGG GGGCGACCGG AGGCCGTCTC GGTGCCCCGC 3120CCGATCAGGC CACTGGGCAC ATCGGGGGCG GGAAGCTGGG CTCACCAAAG GGGCGACTGG 3180CCTTGGCAGG TGTGGGCTCT GGTCCGGCCT GGGCAGGCTC CGGGGGCGGG GTCTCAGGTT 3240ACAGCCCCGC GGGGGGCTGG GGGGCGGCCC GCGGTTTGGG CTGGTTTGCC AGCCTTTGGA 3300GCGACCGGGA GCATATAACC GGAGCCTCTG CTGGGAGAAG ACGCAGAGCG CCGCTGGGCT 3360GCCGGGTCTC CTGCCTCCTC CTCCTGCTCC TAGAGCCTCC TGCATGAGGG CGCGGTAGAG 3420ACCCGGACCC GCTCCGTGCT CTGCCGCCTC GCCGAGCTTC GCCCGCAAGC TGGGGAATTC 3480ATATGTCTAG ATTAGATAAA AGTAAAGTGA TTAACAGCGC ATTAGAGCTG CTTAATGAGG 3540TCGGAATCGA AGGTTTAACA ACCCGTAAAC TCGCCCAGAA GCTAGGTGTA GAGCAGCCTA 3600CATTGTATTG GCATGTAAAA AATAAGCGGG CTTTGCTCGA CGCCTTAGCC ATTGAGATGT 3660TAGATAGGCA CCATACTCAC TTTTGCCCTT TAGAAGGGGA AAGCTGGCAA GATTTTTTAC 3720GTAATAACGC TAAAAGTTTT AGATGTGCTT TACTAAGTCA TCGCGATGGA GCAAAAGTAC 3780ATTTAGGTAC ACGGCCTACA GAAAAACAGT ATGAAACTCT CGAAAATCAA TTAGCCTTTT 3840TATGCCAACA AGGTTTTTCA CTAGAGAATG CATTATATGC ACTCAGCGCT GTGGGGCATT 3900TTACTTTAGG TTGCGTATTG GAAGATCAAG AGCATCAAGT CGCTAAAGAA GAAAGGGAAA 3960CACCTACTAC TGATAGTATG CCGCCATTAT TACGACAAGC TATCGAATTA TTTGATCACC 4020AAGGTGCAGA GCCAGCCTTC TTATTCGGCC TTGAATTGAT CATATGCGGA TTAGAAAAAC 4080AACTTAAATG TGAAAGTGGG TCCGCGTACA GCCGCGCGCG TACGAAAAAC AATTACGGGT 4140CTACCATCGA GGGCCTGCTC GATCTCCCGG ACGACGACGC CCCCGAAGAG GCGGGGCTGG 4200CGGCTCCGCG CCTGTCCTTT CTCCCCGCGG GACACACGCG CAGACTGTCG ACGGCCCCCC 4260CGACCGATGT CAGCCTGGGG GACGAGCTCC ACTTAGACGG CGAGGACGTG GCGATGGCGC 4320ATGCCGACGC GCTAGACGAT TTCGATCTGG ACATGTTGGG GGACGGGGAT TCCCCGGGTC 4380CGGGATTTAC CCCCCACGAC TCCGCCCCCT ACGGCGCTCT GGATATGGCC GACTTCGAGT 4440TTGAGCAGAT GTTTACCGAT GCCCTTGGAA TTGACGAGTA CGGTGGGTAG GGGGCGCGAG 4500GATCCTCTAG AGGGCCCTAT TCTATAGTGT CACCTAAATG CTAGAGGATC TTTGTGAAGG 4560AACCTTACTT CTGTGGTGTG ACATAATTGG ACAAACTACC TACAGAGATT TAAAGCTCTA 4620AGGTAAATAT AAAATTTTTA AGTGTATAAT GTGTTAAACT ACTGATTCTA ATTGTTTGTG 4680TATTTTAGAT TCCAACCTAT GGAACTGATG AATGGGAGCA GTGGTGGAAT GCCTTTAATG 4740AGGAAAACCT GTTTTGCTCA GAAGAAATGC CATCTAGTGA TGATGAGGCT ACTGCTGACT 4800CTCAACATTC TACTCCTCCA AAAAAGAAGA GAAAGGTAGA AGACCCCAAG GACTTTCCTT 4860CAGAATTGCT AAGTTTTTTG AGTCATGCTG TGTTTAGTAA TAGAACTCTT GCTTGCTTTG 4920CTATTTACAC CACAAAGGAA AAAGCTGCAC TGCTATACAA GAAAATTATG GAAAAATATT 4980TGATGTATAG TGCCTTGACT AGAGATCATA ATCAGCCATA CCACATTTGT AGAGGTTTTA 5040CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT GAATGCAATT 5100GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA 5160AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC 5220AATGTATCTT ATCATGTCTG GATCATCCCG CCATGGGTAT ACATCGATGC GGCCGC 5276 7664base pairs nucleic acid single linear cDNA 10 GGTACCACTA GTAAGCTTAGATCCACTGTC TGGGATTATA TCAGGACAAC CGAAGCCTGG 60 AAAGTGTATT AGGTAGAGCATTTTCTTCCA CGTGTTTGGG CACGTTTCCG ACAGCTAGGA 120 TTCCAGCTCT GTCTTTGTATGTTACAGACT GTAAATCAAT CGCAGGTGAA ACTGTTTGGA 180 CAGTAGGTGG GGATCAAAGACCCTCCGCCC GTGAGACTCT AGGCGCTTTC CCCTGCCACC 240 AGCCTGTCTC CAGAGATGCTCTGGAAGGAG GCGGGCCCGG GCGGTCTTTC TGCTCTTTAG 300 CGTGGCGGAC GCGGCGGCGGGGGCAGGGCT GGAGCAGAGA GCGCTGCAGT GATAGAACTT 360 TCTGACCCCG CTGCGCAGGGCGGCAGGGTG GCAGGGTGGC AGGGTGGCGA GCTAAGCCAG 420 AGCCGAACGC TGGAGCTCTGGGAGGAACAT CGAAGTGTTT GTATGTGGTC TGAGATCGGC 480 CTGACTATAT TTTTTTGTCCTAAATTTGCA AGCACACACC CACAAAGCTG CGGTCTTGAC 540 CGGTATTCTT TATAGAGCGCAATGGAGTGA GCTGAGTGTC TAAACGATTT CCCTAATTCA 600 TCTGATAGCA GAGGCGCTCTCCTAATTGGC GAAGAGCTGC CTCATGTCCG CAACTTTTTG 660 GCAGAGTGAA TTCCACAGCTTTGTGTGTGT GTGTGGGGGG GGGTGTAAGG GGTGTCTAAA 720 ACTTTCGGTC TCCTACTATTCTGTATCTCG ACCGGTTGGT TTTACACCCC GGCTCATCTC 780 ATCAACGCAA ACACCCCCACTCTCCTATGG ACCCAAGGAC CTGACGTGGG GGAAGGTGGA 840 CATTAGGAAT GTCAGAAACCTAGAGTCCAC GCTCCTCCTC TCCATCTTTC CACGAGTTTG 900 GGAAACTTCT TGGCTGCGAAGACTTTGACC CACATCTGCA TTTCTCAGCC CCAGCTTCCA 960 AAAGTGCTGC AGGTTCGGGAGGGGAGACCT CAGTCCTCCT TTGTGAGGCT TGTTTGCGTT 1020 GAGGGATTGG CAGCGATGGCTTCCAGATGG GCTGAAACCC TGCCCGTATT TATTTAAACT 1080 GGTTCCTCGT GGAGAGCTGTGAATCGGGCT CTGTATGCGC TCGAGAAAAG CCCCATTCAT 1140 GAGAGGCAAG GCCCAGTGGGTCCCCCCGAC TCCCCGACCC CCCTCTCCCA CAATATATCC 1200 CCCCTCCCTG TGCCCGCCTGCCGCCACCTC CCGGGCTCCG GCCCCGCGCG CAGCGGCGAC 1260 GAAGCAACAC AGTTCCCCGAAAGAGGTAGC TTTTTAATTG GCCAGCCACA AAGAATCACT 1320 TATGCCGCAC GGCGGTAACGAGGGGAACCG GATCGGGCGG CCAGGATGCT ATCTGTGTAG 1380 CCCTTTTCGT GCCACAATTAGGGTGGTGCT GGCTTCCTCC GACCGCACCT AGGCGATCTG 1440 GTTACACTGT TGGCTCCTTTCTTGGGCAGT CATTTAATCC TACTTTTTAC TCTACGAATG 1500 TCTGTCTGAT GGAGGGCTGTGTCCGGAGCC CCATCCACAA AGAGTCAGCC AGCAGCTCTC 1560 ACACCCGGCT GGATCTCATATGGTGCACTC TCAGTACAAT CTGCTCTGAT GCCGCATAGT 1620 TAAGCCAGCC AAGCTAGCTTGCGCAAGCTA GCTTGCGATC CGTAAAAATG TGTGAGAGTT 1680 ACAAAATGTC TTCCGGGCTAAGATCCGACA GCCATGGTCC AAAGAAGACT TCGGCACTGC 1740 AGACTTAAAA CCAGCTTTCTAGCAGAGGCA GAAGGATCTA GAGCCAAAGG CAAAGACTTG 1800 AATAGGCTGG GAAGATGCAAGAATGGCATT TTACATAAAG AACACTCTCT CCTTTTCCAG 1860 CCAGCACACT TGCATAGAAATTAAGTTTTA CACTTGAAGT TCTTTGTTTC CATCCTGAGA 1920 AGCTCCAAAG TCTGAGGTGGTGTGGTATGC TGGGTAATTC TCCCCACCCC CCAACATTCC 1980 CTGGGGGTTC CATGGGGGTAGCTTCTCCCA AGGACTTCCA GCGGCAACAC AGAAATCCCA 2040 CTTCGAGACA AAGGAGTTACTGCTTAAATC AGGCCCTAAT TTCCAAGGTT CCCTTTGCTT 2100 AAAGTTCCCT AGAGGACCATCTCACTTCTA AAGAAAAGGT GTATTCGGGG ACCCATCCTC 2160 AACCTCCTTG TTATGGAAGGAGACTTCGGG AACAGAGCAA GGGCTGAGCC TCCGGCAGTT 2220 TGGGGTAAGG TTGGGGTTGGGGGGAGCAAG GAAGGCAAGT GAGGCTGGAG GCCCAGGGAT 2280 AGGGGAAGAT GTGTGTGTGTGTGTGTGTGT GTGTGTGTGT GTGTGTGTGT CTCGGGGATG 2340 GTGGTGGTGG ACAACTAGGAAACTCTGGCG CTTTCTCCTC CCCTCACAAA ACTGAGTCCA 2400 GCTGGAGCCG CCTCCAGACTCTCTGGCCAG GGCCTCAGAG TGGTCAACAG TCCCTGGCCA 2460 GCGTTGCTCT CTCCAGGCTAAGGGCACCCA CTCCCCTGGA GATTCCTGAA CCTGGGCCAG 2520 GAAGAGCCGA ATTAGACAAGTGTCTCCAAT CCGGCTGCGT GCGGATTTTG TTGCGGTGTC 2580 CCTCGGTTGT CTGCAGTTCCTTTAGTCCCT TCCCTGGCCT GCCCCTTACA CCTCCACACA 2640 GGTCCCCCTC TGTGTAGGAATACACCAGAC CCTCTCTTAG CCACACACAC CTCCAGTCCC 2700 CCGTCTACCT AGATTTTTTTCATAGCTAGT TGGATGGGGG ATGGGTTAGG GAGGCTGGGT 2760 TTGCGAGCCT CCAGGTGGGAGTTCACCGAC AGGTACTCCG CAAAGGAGCT GGAAGGCAGG 2820 TCTGGAAAAC TGTCCCCCAGATTTAGGATT CTGGGCAGCT TCCATCAGCT TATACTTTGG 2880 CTCCCCCGCC CCCTAAACTCCCCATCCCCA CCTTCCTTTC TCCCGTTACT TCGTCCTCCC 2940 TCGCCTTTCC AGCCTTGAGTCTAAAGCTCC ATGCTTATGC CTCTGCAAAC AACCCCCTCC 3000 CTTCTAACCC CAGCAGAACTCCGAGGAAAG GGGCCGGAGG CCCCCCTTCT CGCCTGTGGT 3060 TAGAGGGGGC AGTGTGGCAGTCCCAAGTGG GGGCGACCGG AGGCCGTCTC GGTGCCCCGC 3120 CCGATCAGGC CACTGGGCACATCGGGGGCG GGAAGCTGGG CTCACCAAAG GGGCGACTGG 3180 CCTTGGCAGG TGTGGGCTCTGGTCCGGCCT GGGCAGGCTC CGGGGGCGGG GTCTCAGGTT 3240 ACAGCCCCGC GGGGGGCTGGGGGGCGGCCC GCGGTTTGGG CTGGTTTGCC AGCCTTTGGA 3300 GCGACCGGGA GCATATAACCGGAGCCTCTG CTGGGAGAAG ACGCAGAGCG CCGCTGGGCT 3360 GCCGGGTCTC CTGCCTCCTCCTCCTGCTCC TAGAGCCTCC TGCATGAGGG CGCGGTAGAG 3420 ACCCGGACCC GCTCCGTGCTCTGCCGCCTC GCCGAGCTTC GCCCGCAAGC TGGGGAATTC 3480 GGATCCCCGG GATCGAAAGAGCCTGCTAAA GCAAAAAAGA AGTCACCATG TCGTTTACTT 3540 TGACCAACAA GAACGTGATTTTCGTTGCCG GTCTGGGAGG CATTGGTCTG GACACCAGCA 3600 AGGAGCTGCT CAAGCGCGATCCCGTCGTTT TACAACGTCG TGACTGGGAA AACCCTGGCG 3660 TTACCCAACT TAATCGCCTTGCAGCACATC CCCCTTTCGC CAGCTGGCTT TATAGCGAAG 3720 AGGCCCGCAC CGATCGCCCTTCCCAACAGT TGCGCAGCCT GAATGGCGAA TGGCGCTTTG 3780 CCTGGTTTCC GGCACCAGAAGCGGTGCCGG AAAGCTGGCT GGAGTGCGAT CTTCCTGAGG 3840 CCGATACTGT CGTCGTCCCCTCAAACTGGC AGATGCACGG TTACGATGCG CCCATCTACA 3900 CCAACGTAAC CTATTCCATTACGGTCAATC CGCCGTTTGT TCCCACGGAG AATCCGACGG 3960 GTTGTTACTC GCTCACATTTAATGTTGATG AAAGCTGGCT ACAGGAAGGC CAGACGCGAA 4020 TTATTTTTGA TGGCGTTAACTTGGCGTTTC ATCTGTGGTG CAACGTGCGC TGGGTCGGTT 4080 ACGGCCAGGA CAGTCGTTTGCCGTCTGAAT TTGACCTGAG CGCATTTTTA CGCGCCGGAG 4140 AAAACCGCCT CGCGGTGATGGTGCTGCGTT GGAGTGACGG CAGTTATCTG GAAGATCAGG 4200 ATATGTGGCG GATGAGCGGCATTTTCCGTG ACGTCTCGTT GCTGCATAAA CCGACTACAC 4260 AAATCAGCGA TTTCCATGTTGCCACTCGCT TTAATGATGA TTTCAGCCGC GCTGAACTGG 4320 AGGCTGAAGT TCAGATGTGCGGCGAGTTGC GTGACTACCT ACGGGTAACA GTTTCTTTAT 4380 GGCAGGGTGA AACGCAGGTCGCCAGCGGCA CCGCGCCTTT CGGCGGTGAA ATTATCGATG 4440 AGCGTGGTGG TTATGCCGATCGCGTCACAC TACGTCTGAA CGTCGAAAAC CCGAAACTGT 4500 GGAGCGCCGA AATCCCGAATCTCTATCGTG CGGTGGTTGA ACTGCACACC GCCGACGGCA 4560 CGCTGATTGA AGCAGAAGCCTGCGATGTCG GTTTCCGCGA GGTGCGGATT GAAAATGGTC 4620 TGCTGCTGCT GAACGGCAAGCCGTTGCTGA TTCGAGGCGT TAACCGTCAC GAGCATCATC 4680 CTCTGCATGG TCAGGTCATGGATGAGCAGA CGATGGTGCA GGATATCCTG CTGATGAAGC 4740 AGAACAACTT TAACGCCGTGCGCTGTTCGC ATTATCCGAA CCATCCGCTG TGGTACACGC 4800 TGTGCGACCG CTACGGCCTGTATGTGGTGG ATGAAGCCAA TATTGAAACC CACGGCATGG 4860 TGCCAATGAA TCTGCTGACCGATGATCCGC GCTGGCTACC GGCGATGAGC GAACGCGTAA 4920 CGCGAATGGT GCAGCGCGATCGTAATCACC CGAGTGTGAT CATCTGGTCG CTGGGGAATG 4980 AATCAGGCCA CGGCGCTAATCACGACGCGC TGTATCGCTG GATCAAATCT GTCGATCCTT 5040 CCCGCCCGGT GCAGTATGAAGGCGGCGGAG CCGACACCAC GGCCACCGAT ATTATTTGCC 5100 CGATGTACGC GCGCGTGGATGAAGACCAGC CCTTCCCGGC TGTGCCGAAA TGGTCCATCA 5160 AAAAATGGCT TTCGCTACCTGGAGAGACGC GCCCGCTGAT CCTTTGCGAA TACGCCCACG 5220 CGATGGGTAA CAGTCTTGGCGGTTTCGCTA AATACTGGCA GGCGTTTCGT CAGTATCCCC 5280 GTTTACAGGG CGGCTTCGTCTGGGACTGGG TGGATCAGTC GCTGATTAAA TATGATGAAA 5340 ACGGCAACCC GTGGTCGGCTTACGGCGGTG ATTTTGGCGA TACGCCGAAC CATCGCCAGT 5400 TCTGTATGAA CGGTCTGGTCTTTGCCGACC GCACGCCGCA TCCAGCGCTG ACGGAAGCAA 5460 AACACCAGCA GCAGTTTTTCCAGTTCCGTT TATCCGGGCA AACCATCGAA GTGACCAGCG 5520 AATACCTGTT CCGTCATAGCGATAACGAGC TCCTGCACTG GATGGTGGCG CTGGATGGTA 5580 AGCCGCTGGC AAGCGGTGAAGTGCCTCTGG ATGTCGCTCC ACAAGGTAAA CAGTTGATTG 5640 AACTGCCTGA ACTACCGCAGCCGGAGAGCG CCGGGCAACT CTGGCTCACA GTACGCGTAG 5700 TGCAACCGAA CGCGACCGGATGGTCAGAAG CCGGGCACAT CAGCGCCTGG CAGCAGTGGC 5760 GTCTGGCGGA AAACCTCAGTGTGACGCTCC CCGCCGCGTC CCACGCCATC CCGCATCTGA 5820 CCACCAGCGA AATGGATTTTTGCATCGAGC TGGGTAATAA GCGTTGGCAA TTTAACCGCC 5880 AGTCAGGCTT TCTTTCACAGCTGTGGATTG GCGATAAAAA ACAACTGCTG ACGCCGCTGC 5940 GCGATCAGTT CACCCGTGCACCGCTGGATA ACGACATTGG CGTAAGTGAA GCGACCCGCA 6000 TTGACCCTAA CGCCTGGGTCGAACGCTGGA AGGCGGCGGG CCATTACCAG GCCGAAGCAG 6060 CGTTGTTGCA GTGCACGGCAGATACACTTG CTGATGCGGT GCTGATTACG ACCGCTCACG 6120 CGTGGCAGCA TCAGGGGAAAACCTTATTTA TCAGCCGGAA AACCTACCGG ATTGATGGTA 6180 GTGGTCAAAT GGCGATTACCGTTGATGTTG AAGTGGCGAG CGATACACCG CATCCGGCGC 6240 GGATTGGCCT GAACTGCCAGCTGGCGCAGG TAGCAGAGCG GGTAAACTGG CTCGGATTAG 6300 GGCCGCAAGA AAACTATCCCGACCGCCTTA CTGCCGCCTG TTTTGACCGC TGGGATCTGC 6360 CATTGTCAGA CATGTATACCCCGTACGTCT TCCCGAGCGA AAACGGTCTG CGCTGCGGGA 6420 CGCGCGAATT GAATTATGGCCCACACCAGT GGCGCGGCGA CTTCCAGTTC AACATCAGCC 6480 GCTACAGTCA ACAGCAACTGATGGAAACCA GCCATCGCCA TCTGCTGCAC GCGGAAGAAG 6540 GCACATGGCT GAATATCGACGGTTTCCATA TGGGGATTGG TGGCGACGAC TCCTGGAGCC 6600 CGTCAGTATC GGCGGAATTACAGCTGAGCG CCGGTCGCTA CCATTACCAG TTGGTCTGGT 6660 GTCAAAAATA ATAATAACCGGCAGGCCATG TCTGAAAGTA TTCGCGTAAG GAAATCCATT 6720 ATGTACTATT TAAAAAACACAAACTTTTGG ATGTTCGGTT TATTCTTTTT CTTTTACTTT 6780 TTTATCATGG GAGCCTACTTCCCGTTTTTC CCGATTTGGC TACATGACAT CAACCATATG 6840 AGCAAAAGTG ATACGGGTATTATTTTTGCC GCTATTTCTC TGTTGTCGCT ATTATTCCAA 6900 CCGCTGTTGG TCTGCTTTCTGACAAACTCG GCCTCGACTC TAGACTGAGA ACTTCAGGGT 6960 GAGTTTGGGG ACCCTTGATTGTTCTTTCTT TTTCGCTATT GAAAAATTCA TGTTATATGG 7020 AGGGGGCAAA GTTTTCAGGGTGTTGTTTAG AATGGGAAGA TGTCCCTTGT ATCACCATGG 7080 ACCCTCATGA TAATTTTGTTTCTTTCACTT TCTACTCTGT TGACAACCAT TGTCTCCTCT 7140 TATTTTCTTT TCATTTTCTGTAACTTTTTT CGTTAAACTT TAGCTTGCAT TTGTAACGAA 7200 TTTTTAAATT CACTTTCGTTTATTTGTCAG ATTGTAAGTA CTTTCTCTAA TCACTTTTTT 7260 TTCAAGGCAA TCAGGGTAATTATATTGTAC TTCAGCACAG TTTTAGAGAA CAATTGTTAT 7320 AATTAAATGA TAAGGTAGAATATTTCTGCA TATAAATTCT GGCTGGCGTG GAAATATTCT 7380 TATTGGTAGA AACAACTACATCCTGGTAAT CATCCTGCCT TTCTCTTTAT GGTTACAATG 7440 ATATACACTG TTTGAGATGAGGATAAAATA CTCTGAGTCC AAACCGGGCC CCTCTGCTAA 7500 CCATGTTCAT GCCTTCTTCTTTTTCCTACA GCTCCTGGGC AACGTGCTGG TTGTTGTGCT 7560 GTCTCATCAT TTTGGCAAAGAATTCACTCC TCAGGTGCAG GCTGCCTATC AGAAGGTGGT 7620 GGCTGGTGTG GCCAATGCCCTGGCTCACAA ATACCACTGA GATC 7664 20 base pairs nucleic acid single linear11 CGAGGGCCTG CTCGATCTCC 20 20 base pairs nucleic acid single linear 12GGCATTCCAC CACTGCTCCC 20 21 base pairs nucleic acid single linear 13GAGCACCCTT CTCATGACCT C 21 22 base pairs nucleic acid single linear 14GTTGGTGTAG ATGGGCGCAT CG 22 21 base pairs nucleic acid single linear 15GCGGGGTCTC AGGTTACAGC C 21 23 base pairs nucleic acid single linear 16GCCCTCTGGC CTGCTGGCTC ATG 23 24 base pairs nucleic acid single linearcDNA 17 CAGGAGAGTC TTGCCTGTAT CCTC 24 1521 base pairs nucleic acidsingle linear cDNA 18 CAAGATGCAT CCAGGGGTCC TGGCTGCCTT CCTCTTCTTGAGCTGGACTC ATTGTCGGGC 60 CCTGCCCCTT CCCAGTGGTG GTGATGAAGA TGATTTGTCTGAGGAAGACC TCCAGTTTGC 120 AGAGCGCTAC CTGAGATCAT ACTACCATCC TACAAATCTCGCGGGAATCC TGAAGGAGAA 180 TGCAGCAAGC TCCATGACTG AGAGGCTCCG AGAAATGCAGTCTTTCTTCG GCTTAGAGGT 240 GACTGGCAAA CTTGACGATA ACACCTTAGA TGTCATGAAAAAGCCAAGAT GCGGGGTTGT 300 CGATGTGGG TGAATACAATG TTTTCCCTCG AACTCTTAAATGGTCCAAAA TGAATTTAAC 360 CTACAGAATT GTGAATTACA CCCCTGATAT GACTCATTCTGAAGTCGAAA AGGCATTCAA 420 AAAAGCCTTC AAAGTTTGGT CCGATGTAAC TCCTCTGAATTTTACCAGAC TTCACGATGG 480 CATTGCTGAC ATCATGATCT CTTTTGGAAT TAAGGAGCATGGCGACTTCT ACCCATTTGA 540 TGGGCCCTCT GGCCTGCTGG CTCATGCTTT TCCTCCTGGGCCAAATTATG GAGGAGATGC 600 CCATTTTGAT GATGATGAAA CCTGGACAAG TAGTTCCAAAGGCTACAACT TGTTTCTTGT 660 TGCTGCGCAT GAGTTCGGCC ACTCCTTAGG TCTTGACCACTCCAAGGACC CTGGAGCACT 720 CATGTTTCCT ATCTACACCT ACACCGGCAA AAGCCACTTTATGCTTCCTG ATGACGATGT 780 ACAAGGGATC CAGTCTCTCT ATGGTCCAGG AGATGAAGACCCCAACCCTA AACATCCAAA 840 AACGCCAGAC AAATGTGACC CTTCCTTATC CCTTGATGCCATTACCAGTC TCCGAGGAGA 900 AACAATGATC TTTAAAGACA GATTCTTCTG GCGCCTGCATCCTCAGCAGG TTGATGCGGA 960 GCTGTTTTTA ACGAAATCAT TTTGGCCAGA ACTTCCCAACCGTATTGATG CTGCATATGA 1020 GCACCCTTCT CATGACCTCA TCTTCATCTT CAGAGGTAGAAAATTTTGGG CTCTTAATGG 1080 TTATGACATT CTGGAAGGTT ATCCCAAAAA AATATCTGAACTGGGTCTTC CAAAAGAAGT 1140 TAAGAAGATA AGTGCAGCTG TTCACTTTGA GGATACAGGCAAGACTCTCC TGTTCTCAGG 1200 AAACCAGGTC TGGAGATATG ATGATACTAA CCATATTATGGATAAAGACT ATCCGAGACT 1260 AATAGAAGAA GACTTCCCAG GAATTGGTGA TAAAGTAGATGCTGTCTATG AGAAAAATGG 1320 TTATATCTAT TTTTTCAACG GACCCATACA GTTTGAATACAGCATCTGGA GTAACCGTAT 1380 TGTTCGCGTC ATGCCAGCAA ATTCCATTTT GTGGTGTTAAGTGTCTTTTT AAAAATTGTT 1440 ATTTAAATCC TGAAGAGCAT TTGGGGTAAT ACTTCCAGAAGTGCGGGGTA GGGGAAGAAG 1500 AGCTATCAGG AGAAAGCTTG G 1521 7 amino acidsamino acid single linear protein 19 Pro Arg Cys Gly Xaa Pro Asp 1 5 12amino acids amino acid single linear protein 20 His Glu Xaa Gly His XaaXaa Xaa Xaa Xaa His Ser 1 5 10 471 amino acids amino acid single linearprotein 21 Met His Pro Gly Val Leu Ala Ala Phe Leu Phe Leu Ser Trp ThrHis 1 5 10 15 Cys Arg Ala Leu Pro Leu Pro Ser Gly Gly Asp Glu Asp AspLeu Ser 20 25 30 Glu Glu Asp Leu Gln Phe Ala Glu Arg Tyr Leu Arg Ser TyrTyr His 35 40 45 Pro Thr Asn Leu Ala Gly Ile Leu Lys Glu Asn Ala Ala SerSer Met 50 55 60 Thr Glu Arg Leu Arg Glu Met Gln Ser Phe Phe Gly Leu GluVal Thr 65 70 75 80 Gly Lys Leu Asp Asp Asn Thr Leu Asp Val Met Lys LysPro Arg Cys 85 90 95 Gly Gly Val Asp Val Gly Glu Tyr Asn Val Phe Pro ArgThr Leu Lys 100 105 110 Trp Ser Lys Met Asn Leu Thr Tyr Arg Ile Val AsnTyr Thr Pro Asp 115 120 125 Met Thr His Ser Glu Val Glu Lys Ala Phe LysLys Ala Phe Lys Val 130 135 140 Trp Ser Asp Val Thr Pro Leu Asn Phe ThrArg Leu His Asp Gly Ile 145 150 155 160 Ala Asp Ile Met Ile Ser Phe GlyIle Lys Glu His Gly Asp Phe Tyr 165 170 175 Pro Phe Asp Gly Pro Ser GlyLeu Leu Ala His Ala Phe Pro Pro Gly 180 185 190 Pro Asn Tyr Gly Gly AspAla His Phe Asp Asp Asp Glu Thr Trp Thr 195 200 205 Ser Ser Ser Lys GlyTyr Asn Leu Phe Leu Val Ala Ala His Glu Phe 210 215 220 Gly His Ser LeuGly Leu Asp His Ser Lys Asp Pro Gly Ala Leu Met 225 230 235 240 Phe ProIle Tyr Thr Tyr Thr Gly Lys Ser His Phe Met Leu Pro Asp 245 250 255 AspAsp Val Gln Gly Ile Gln Ser Leu Tyr Gly Pro Gly Asp Glu Asp 260 265 270Pro Asn Pro Lys His Pro Lys Thr Pro Asp Lys Cys Asp Pro Ser Leu 275 280285 Ser Leu Asp Ala Ile Thr Ser Leu Arg Gly Glu Thr Met Ile Phe Lys 290295 300 Asp Arg Phe Phe Trp Arg Leu His Pro Gln Gln Val Asp Ala Glu Leu305 310 315 320 Phe Leu Thr Lys Ser Phe Trp Pro Glu Leu Pro Asn Arg IleAsp Ala 325 330 335 Ala Tyr Glu His Pro Ser His Asp Leu Ile Phe Ile PheArg Gly Arg 340 345 350 Lys Phe Trp Ala Leu Asn Gly Tyr Asp Ile Leu GluGly Tyr Pro Lys 355 360 365 Lys Ile Ser Glu Leu Gly Leu Pro Lys Glu ValLys Lys Ile Ser Ala 370 375 380 Ala Val His Phe Glu Asp Thr Gly Lys ThrLeu Leu Phe Ser Gly Asn 385 390 395 400 Gln Val Trp Arg Tyr Asp Asp ThrAsn His Ile Met Asp Lys Asp Tyr 405 410 415 Pro Arg Leu Ile Glu Glu AspPhe Pro Gly Ile Gly Asp Lys Val Asp 420 425 430 Ala Val Tyr Glu Lys AsnGly Tyr Ile Tyr Phe Phe Asn Gly Pro Ile 435 440 445 Gln Phe Glu Tyr SerIle Trp Ser Asn Arg Ile Val Arg Val Met Pro 450 455 460 Ala Asn Ser IleLeu Trp Cys 465 470

What is claimed is:
 1. A transgenic mouse whose genome comprises: (a) anucleotide sequence encoding a constitutively enzymatically active humanmatrix metalloproteinase that cleaves Type II collagen, wherein thenucleotide sequence encoding the metalloproteinase is operatively linkedto a regulatable promoter; and (b) a nucleotide sequence encoding arepressor-activator fusion polypeptide that binds to the regulatablepromoter in the absence of a repressor-activator fusionpolypeptide-binding compound and does not bind to the regulatablepromoter in the presence of the compound, which nucleotide sequenceencoding the repressor-activator fusion polypeptide is operativelylinked to a Type II collagen promoter, wherein expression of themetalloproteinase is capable of being repressed in the mouse untiladulthood, and wherein the metalloproteinase is capable of beingexpressed in the mouse during adulthood to a level sufficient to causeType II collagen degradation in the joints of the mouse.
 2. Thetransgenic mouse of claim 1, wherein the matrix metalloproteinase isselected from the group consisting of MMP-1, MMP-8, and MMP-13.
 3. Thetransgenic mouse of claim 2, wherein the matrix metalloproteinase isMMP-13.
 4. The transgenic mouse of claim 3, wherein the MMP-13 comprisesthe sequence of SEQ ID NO:1 or SEQ ID NO:21.
 5. The transgenic mouse ofclaim 1, wherein the repressor-activator fusion polypeptide is achimeric tetracycline repressor-VP16 transcription activator polypeptideand the regulatable promoter is a Tn10-sequence linked to a portion ofthe CMV IE promoter.
 6. The transgenic mouse of claim 5, wherein theregulatable promoter comprises the sequence of SEQ ID NO:2.
 7. Thetransgenic mouse of claim 1, wherein the Type II collagen degradationresults in a loss of proteoglycan, cleavage of type II collagen into aTC^(A) degradation product, a change in joint function, joint spacenarrowing, destruction of cartilage, a change in growth platemorphology, fibrillation and loss of articular cartilage, osteophyteformation, or combinations thereof.
 8. A transgenic mouse whose genomecomprises: (a) a nucleotide sequence encoding a constitutivelyenzymatically active human matrix metalloproteinase that cleaves Type IIcollagen, wherein the nucleotide sequence encoding the metalloproteinaseis operatively linked to a tetracycline-regulatable promoter; and (b) anucleotide sequence encoding a repressor-activator fusion polypeptidethat binds to the tetracycline regulatable promoter in the absence oftetracycline or a tetracycline analog and does not bind to theregulatable promoter in the presence of tetracycline or tetracyclineanalog, which nucleotide sequence encoding the repressor-activatorfusion polypeptide is operatively linked to a Type II collagen promoter,wherein expression of the metalloproteinase is capable of beingrepressed in the mouse until adulthood, and wherein themetalloproteinase is capable of being expressed in the mouse duringadulthood to a level sufficient to cause Type II collagen degradation inthe joints of the mouse.
 9. The transgenic mouse of claim 8, wherein thematrix metalloproteinase is constitutively enzymatically active MMP-13,the tetracycline-regulatable promoter is tet07, and therepressor-activator fusion polypeptide is tTA.
 10. The transgenic mouseof claim 8, wherein the Type II collagen degradation results in a lossof proleoglycan, cleavage of type II collagen into a TC^(A) degradationproduct, a change in joint function, joint space narrowing, destructionof cartilage, a change in growth plate morphology, fibrillation and lossof articular cartilage, osteophyte formation, or combinations thereof.11. A method for producing degradation of Type II collagen in the jointsof a transgenic mouse, which method comprises: (a) maintaining thetransgenic mouse of claim 1 in presence of the transcription activatorprotein-binding compound until adulthood; and (b) activating expressionof the matrix metalloproteinase in the transgenic mouse by withholdingthe compound from the mouse after the mouse has reached adulthood suchthat the matrix metalloproteinase degrades Type II collagen in thejoints of the transgenic mouse.
 12. The method according to claim 11,wherein the Type II collagen degradation results in a loss ofproteoglycan, cleavage of type II collagen into a TC^(A) degradationproduct, a change in joint function, joint space narrowing, destructionof cartilage, a change in growth plate morphology, fibrillation and lossof articular cartilage, osteophyte formation, or combinations thereof.13. A method for producing degradation of Type II collagen in the jointsof a transgenic mouse, which method comprises: (a) maintaining thetransgenic mouse of claim 11 in the presence of tetracycline or atetracycline analog until adulthood; and (b) activating expression ofthe matrix metalloproteinase by withholding the tetracycline ortetracycline analog from the mouse after the mouse has reachedadulthood, such that the matrix metalloproteinase degrades Type IIcollagen in the joints of the transgenic mouse.
 14. The method accordingto claim 13, wherein the tetracycline analog is doxycycline.
 15. Themethod according to claim 13, wherein the Type II collagen degradationresults in a loss of proteoglycan, cleavage of type II collagen into aTC^(A) degradation product, a change in joint function, joint spacenarrowing, destruction of cartilage, a change in growth platemorphology, fibrillation and loss of articular cartilage, osteophyteformation, or combinations thereof.
 16. A method for producingdegradation of Type II collagen in the joints of a transgenic mouse,which method comprises (a) maintaining the transgenic mouse of claim 8in the presence of tetracycline or a tetracycline analog untiladulthood; and (b) activating expression of the matrix metalloproteinaseby withholding the tetracycline or tetracycline analog from the mouseafter the mouse has reached adulthood, such that the matrixmetalloproteinase degrades Type II collagen in the joints of thetransgenic mouse.
 17. The method according to claim 16, wherein thetetracycline analog is doxycycline.
 18. The method according to claim16, wherein the Type II collagen degradation results in a loss ofproteoglycan, cleavage of type II collagen into a TC^(A) degradationproduct, a change in joint function, joint space narrowing, destructionof cartilage, a change in growth plate morphology, fibrillation and lossof articular cartilage, osteophyte formation, or combinations thereof.19. A transgenic mouse whose genome comprises: (a) a nucleotide sequenceencoding a constitutively enzymatically active human matrixmetalloproteinase that cleaves Type II collagen, wherein the nucleotidesequence encoding the metalloproteinase is operatively linked to aregulatable promoter; and (b) a nucleotide sequence encoding atranscription activator protein that binds to the regulatable promoterin the presence of a transcription activator protein-binding compoundand does not bind to the regulatable promoter in the absence of thecompound, which nucleotide sequence encoding the transcription activatorprotein is operatively linked to a Type II collagen promoter; whereinexpression of the metalloproteinase is capable of being repressed in themouse until adulthood, and wherein the metalloproteinase is capable ofbeing expressed in the mouse during adulthood to a level sufficient tocause Type II collagen degradation in the joints of the mouse.
 20. Thetransgenic mouse of claim 19, wherein the matrix metalloproteinase isselected from the group consisting of MMP-1, MMP-8, and MMP-13.
 21. Thetransgenic mouse of claim 20, wherein the matrix metalloproteinase isMMP-13.
 22. The transgenic mouse of claim 21, wherein the MMP-13comprises the sequence of SEQ ID NO:1 or SEQ ID NO:21.
 23. Thetransgenic mouse of claim 19, wherein the transcription activatorprotein is a chimeric polypeptide comprising a transactivator domainlinked to an ecdysone receptor ligand-binding domain, and wherein thetransgenic mouse further comprises a nucleotide sequence encoding aretinoid X receptor (RXR), which nucleotide sequence encoding RXR isoperatively linked to a Type II collagen promoter.
 24. The transgenicmouse of claim 19, wherein the transcription activator protein is achimeric polypeptide comprising a transactivator domain linked to aprogesterone receptor ligand-binding domain.
 25. The transgenic mouse ofclaim 19, wherein the transcription activator protein is a chimericpolypeptide comprising a transactivator domain linked to a steroidbinding domain.
 26. The transgenic mouse of claim 19, wherein the TypeII collagen degradation results in a loss of proteoglycan, cleavage oftype II collagen into a TC^(A) degradation product, a change in jointfunction, joint space narrowing, destruction of cartilage, a change ingrowth plate morphology, fibrillation and loss of articular cartilage,osteophyte formation, or combinations thereof.
 27. A method forproducing degradation of Type II collagen in the joints of a transgenicmouse, which method comprises: (a) maintaining the transgenic mouse ofclaim 19 in the absence of the transcription activator protein-bindingcompound until adulthood; and (b) activating expression of the matrixmetalloproteinase in the transgenic mouse by administering the compoundto the mouse after the mouse has reached adulthood such that the matrixmetalloproteinase degrades Type II collagen in the joints of the mouse.28. A method for producing degradation of Type II collagen in the jointsof a transgenic mouse, which method comprises: (a) maintaining thetransgenic mouse of claim 23 in the absence of ecdysone, an ecdysoneanalog, or dexamethasone until adulthood; and (b) activating expressionof the matrix metalloproteinase in the transgenic mouse by administeringecdysone, an ecdysone analog, or dexamethasone to the mouse after themouse has reached adulthood such that the matrix metalloproteinasedegrades Type II collagen in the joints of the mouse.
 29. A method forproducing degradation of Type II collagen in the joints of a transgenicmouse, which method comprises: (a) maintaining the transgenic mouse ofclaim 24 in the absence of mifeprestone (RU 486) until adulthood; and(b) activating expression of the matrix metalloproteinase in thetransgenic mouse by administering mifepristone (RU 486) to the mouseafter the mouse has reached adulthood such that the matrixmetalloproteinase degrades Type II collagen in the joints of the mouse.30. The method according to claim 28, wherein the Type II collagendegradation results in a loss of proteoglycan, cleavage of type IIcollagen into a TC^(A) degradation product, a change in joint function,joint space narrowing, destruction of cartilage, a change in growthplate morphology, fibrillation and loss of articular cartilage,osteophyte formation, or combinations thereof.
 31. The method accordingto claim 29, wherein the Type II collagen degradation results in a lossof proteoglycan, cleavage of type II collagen into a TC^(A) degradationproduct, a change in joint function, joint space narrowing, destructionof cartilage, a change in growth plate morphology, fibrillation and lossof articular cartilage, osteophyte formation, or combinations thereof.